Effects of Photoperiod on Mammary Development and Concentration of Hormones in Serum of Pregnant Dairy Heifers

PHYSIOLOGY AND MANAGEMENT Effects of Photoperiod on Mammary Development and Concentration of Hormones in Serum of Pregnant Dairy Heifers L. T.

CHAPIN, S. A. 21": and H. ALLEN TUCKER2 Department of Animal Science Michigan State University East Lansing 48824

J. A. NEWBOLD,

ABSTRACT

Abbreviation key: L = light, D = darkness.

Beginning at 128 d of pregnancy, Holstein heifers were exposed to 16 h light, 8 h dark (long days; n = 10) or 8 h light, 16 h dark (short days; n = 10) until 35 d before calving when they were killed. photoperiod had no effect on weight or proportion of extraparenchymal fat or parenchyma in the mammary gland or on amount of fat, DNA, or RNA in mammary parenchyma. Serum prolactin concentration was 1.7-fold greater under long than under short days. Concentration of melatonin in serum was 2.4-fold greater during dark than light periods. Duration of elevated serum melatonin concentration in the dark period was longer in heifers given short days, but magnitude of this increase was lower than that in heifers exposed to long days. In a second experiment, peak amplitude and area of the periparturient surge of serum concentmtion of prolactin were 1.8-fold and 1.7-fold greater, respectively, in six Holstein heifers exposed to long days than in six heifers exposed to short days. We conclude that photoperiod had no effect on mammary development during pregnancy, but relative to short days, long days increased serum concentration of prolactin during pregnancy, including the period of the periparturient surge of prolactin. (Key words: photoperiod, pregnancy, mammary development)

INTRODUCTION

Increasing the number or activity of mammary epithelial cells stimulates milk yield. In

previous studies, increasing daily exposure of light from 8 to 16 h increased the total amount of mammary parenchymal DNA (an index of cell number) in the allometric growth phases of the mammary gland of prepubertal and postpubertal, nonpregnant heifers (16). Although most allometric mammary growth occurs during pregnancy (24), effects of photoperiod on mammary growth and on concentrations of prolactin and progesterone in serum, key hormones involved in mammary gland development during pregnancy (22,26), have not been reported. photoperiod markedly affects serum concentration of melatonin in many species, including bull calves (19), and exogenous melatonin suppresses mammary growth in prepubertal heifers (Sanchez-Biucelo et al., 1991, unpublished data). Lactogenesis occurs concurrently with mammary development during late pregnancy, and a periparturient surge in concentration of serum prolactin is required for maximal stimulation of lactogenesis. For example, blocking the periparhnient surge of prolactin in serum reduces milk yield in dairy cows by 11 kg/d during the first 10 d of lactation (1). However, effects of photoperiod on the penparturient surge of prolactin in serum have not been studied. Our first objective was to determine the effect of photoperiod on mammary development and serum concentration of prolactin, proReceived March 10, 1990. gesterone, and melatonin during pregnancy. Accepted Augnst 7,1990. Our second objective was to determine the 'Present addre~s:Del#utment of ~nimalScience, unieffect of photoperiod on prolactin concentration vcrSity of Cormecticut, Storrs 06269. 2Rcprint requests. in serum during the periparturient period. 1991 J Dairy Sci 74100-108

100

PHOTOPEIUOD EpFEcrrS DURING E'REGNANCY

MATERIALS AND METHODS

Experiment 1 Treatments and Management. Holstein heifers were divided into five blocks of four heifers each according to stage of pregnancy (calculated from breeding date). To acclimate heifers to short days, heifers were exposed to a photoperiod of 8 h of light and 16 h of darhess (8k 16D) until 128 d of pregnancy. Because mean stage of pgnancy differed between blocks, the duration of the acclimation period ranged from 28 to 80 d At 128 d of pregnancy, heifers were assigned randomly from within a block to one of four pens. From 128 to 248 f 1.1 d of pregnancy, two pens of heifers were switched to a treatment photoperiod of 16 h of light and 8 h of darkness (16L:8D), and two pens continued to receive 8L16D (10 heifers per photope nod). Lights came on at 0700 h each day in all pens and provided an intensity of 298 f 9 lx, measured at 22 sites throughout each pen at 1 m from the floor. Heifers were bedded on straw and fed, on a wet weight basis, a complete mixed diet of 68% corn silage, 27% alfalfa haylage, 5% soybean meal and .3% vitamin-mineral supplement at the rate of 9 kg DWd per heifer. The diet was formulated to support .7 kg live weight gain/d. Heifers were weighed b e f m feeding on 3 consecutive d at the Start (128 d of pregnancy) and end of the experiment. Heifers were transported to an abattoir and killed (stunning gun followed by exsanguination) at 248 f 1.1 d of pregnancy. Two heifers (one from each light treatment) aborted, and their data were deleted from the experiment. Blood Sampling. Blood was collected over 24-h periods at three different times during the experiment beginning June 1 and ending July 30. The average day of gestation at the time of bleeding was 160,197, and 219 d with a range of f 26 d of gestation. An indwelling catheter was inserted aseptically in a jugular vein on the day before blood collection. Beginning at 0700 h on the following day, blood samples (2 ml) were drawn and discarded at 20-min intervals for 2 h to accustom heifers to the sampling procedure. Samples of blood (10 ml) were then collected every 30 min for 6 h for measurement of prolactin Cancentration, every 2 h for 24 h for melatonin concentration, and every 8 h for

101

24 h for progesterone concentration. A red light ( 4 1 Ix at a distance of .5 m) was used to aid in collection of blood during the dark period. A p proximately 1 min was required to collect each sample. Heifers received no light during the dark periods except on the days of blood samp ling. Blood samples were stored 2 to 4 h at approximately 25'C and then for 15 h at 4'C. After centrifugation, serum was decanted and stored at -20'C until assayed for concentrations of prolactin [(ll); see Appendix], melatonin (20, 27), and progesterone (18). The prolactin standard was NIH-bPRL-BA Melatonin and progesterone standards were from Sigma Chemical Co. (St. Louis, MO). Interassay and inmassay coefficients of variation were 16.8 and 10.0% for prolactin, 9.5 and 11.0% for melatonin, and 7.5 and 8.3% for progesterone. Mammary Gland and Carcass Analyses. After slaughter, the mammary gland was removed, weighed, and two parenchymal tissue samples were collected from the right rear quarter for histological examination (2). These samples were fixed (9), dehydrated, embedded in JB-4 plus resin (Polysciences Inc., Warrington, PA), sliced into 2-pm sections, and stained with toluidine blue. Four sections from each heifer were examined at a magnification of 4OOx Stromal tissue, epithelial cells, and alveolar lumen were identified and counted at 64 intersections of an ocular planimeter for three fields of view per section chosen at random. This procedure gave 768 observations for each animal.

Immediately after collection of the histological samples, the mammary glands were frozen and stored at -2o'C. Subsequently, the mammary glands were sliced into 10-mm sections, lymph and skin, teats, and suprnodes were removed. Each slice was dissected into extraparenchymal fat and parenchyma Parenchymal tissue was pooled across slices, weighed, and analyzed for DNA, RNA (23), and ether-extracted fat (3). At slaughter, the fetus was removed from placental membranes, and weight and crown to rump length were determined. Weight of carcass and perirenal fat were measured at slaughter. Depth of subcutanmus fat over the 12th rib and area of longissimus dorsi muscle between the 12th and 13th rib were measured on the chilled carcass 24 h after slaughter. Journal of Dairy Science Vol. 74. No. 1, 1991

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NEWBOLD ET AL.

Statistical Analysis. Differences in mammary gland composition, live weight gain, and

data obtained at the time of slaughter were tested for significance by analysis of variance (5). When significant (P<.l), initial BW was included in the model as a covariate for the indices of mammary development and carcass and fetal characteristics. Sex of fetus was tested as a covariate for fetal characteristics, but it was not significant. Differences in prolactin, melatonin, and progesterone concentrations of serum were analyzed using split-plot analysis with repeated measurements over time (6). Concentration and area of melatonin in serum during light and dark periods within each day were compared using the Bonferroni t test (6). Stage of pregnancy at the time of first blood sampling (based on breeding dates and confirmed by fetal crown to rump length) was tested as a covariate in the statistical analyses of hormone concentrations. Ambient temperature during collection of each blood sample was also tested as a covariate in the statistical analysis of prolactin concentrations. Experiment 2 Treatments and Management. Twelve Holstein heifers (artificially inseminated on same date after synchronization of estrous cycles with prostaglandin-F& were assigned randomly to one of four pens 227 d after breeding, which corresponds to 56 d before expected calving date. All heifers were exposed to 8 L 16D for a 21 d pretreatment period. Beginning at 35 d before expected date of parturition, heifers in two pens received 16L:8D, although the other two pens remained on 8L:16D. Light intensities and diets were similar to those of Experiment 1. Animals remained on treatment until approximately 2 h after parturition, at which time they were separated from their calves and moved 85 m to another barn where the heifers received continuous lighting at 1103 f 170 Ix. Two animals, one on each treatment, calved either during or immediately after the first 48 h of blood collection; prolactin concentration of these animals was not used to calculate the basal concentration of prolactin in se rum. However, data from all heifers were used to characterize the penparturient surge of prolactin. J o d of Dairy Science Vol. 74. No. 1, 1991

Blood Sampling. On March 31, 14 d before expected calving date (269 d of pregnancy), an indwelling catheter was inserted aseptically into a jugular vein of each heifer. Beginning the following morning at 0700 h, blood samples were collected at 2-h intervals for 48 h. Prolactin and melatonin concentrations in serum during this period (defined as basal concentration) were measured. Thereafter, blood samples were collected at 441intervals until 48 h after parturition. Serum samples from 72 h before to 48 h after parturition were assayed for prolactin as in Experiment 1. Statistical Analysis. Basal concentrations of prolactin and melatonin in serum were analyzed as in Experiment 1. prolactin concentration in serum from 72 h before to 48 h after parturition was used to define the penparturient prolactin surge. A pulse analysis computer program [PULSAR; (13)] was used to define the beginning and end points of the prolactin surge. Once duration of the surge was defined, the average concentration of prolactin in serum for the 72 h prior to the surge was defined as the presurge concentration. The increase in serum concentration of prolactin above the presurge serum concentration was defined as the area under the prolactin surge. The area of the surge was calculated using the trapezoidal rule for both total duration of the surge (as defined by the PULSAR program) and from the beginning of the surge until parturition. The postsurge concentration of prolactin included those values between the end of the surge and 48 h after parturition. Defined characteristics of the prolactin secretory pattern included. presurge and postsurge concentrations, area of the surge, peak senun concentration (maximum serum concentration attained during the surge), and peak amplitude (surge peak minus presurge serum concentration). These characteristics were analyzed by analysis of variance (5). RESULTS

Experiment 1

Mammary Development. There was no effect of photoperiod (1&8D vs. 8L16D) on total mammary gland weight (includes parenchyma, extraparenchymal fat, teats, skin, and supramammary lymph nodes), total mammary parenchymal weight, total extraparenchymal fat

103

PHOTOPBRIOD EFFECTS DURING PREGNANCY

TABLE 1. Effect of photoperiod on gross time composition of the mannnary gland in pregnant heifers. Treatment Mammary gLand weight, kg Exmpzua~chymalfat

4?

46 Gland weight

parenchyma

kg 46 Gland weight

16L:8D1

8L1d

SE3

P

11.39

14.0

1.05

.12

3.15 34A

4.44 40.4

.I53 3.30

544 53

6.40 65.6

728 59.6

.99

.44

3.30

53

'Sixteen horns of light and 8 h of darkness. 2Eight hours of light and 16 h of dadolass. 3PoOled SE. 4~obabilityvalac ~ ~ j w t efor t i significant covariate (initial BW). Treatment meam were unadjusted.

weight, or proportion of parenchyma or extrapmchyma in the mammary gland flable 1). photoperiod treatment had no effect on DM cancentration, etherextracted fat concentration, total amount or concentration of nucleic acids, or relative amounts of stromal tissue, epithelial cells, and alveolar lumen in the parenchyma (Table 2). Heifer Growth, Carcass,and Feral Analyses. Initial BW of heifers assigned to 16L:8D was less than that of heifers maintained on 8L:16D (P<.O2; Table 3). However, photoperiod did not significantly affect final B W or live weight gain, carcass weight, perirenal fat weight, subcutaneous fat depth, longissimus dorsi area of

heifers or fetal crown to rump length or fetal weight (Table 3). Serum Hormones. Stage of pregnancy was not a significant (f5.10) covariate in the analyses of serum hormone Concentrations. Averaged across a l l 3 d of pregnancy, photoperid did not influence serum progesterone (mean = 3.6 f .3 ng/ml for 16L:SD and 3.8 f .3 n g / d for 8 L 16D. P = S6). Mean prolactin in serum (pooled across the 3 d of pregnancy) was greater (P = .02)for heifers exposed to 16L:8D (104 f 9 ng/ ml) than for heifers exposed to 8L16D (62 f 9 ng/ml). Prolactin concentration in serum increased as pregnancy advanced from 160 to 219 d (P = .04;Figure 1). Although average ambient temperam increased from 160 to 219 d,

TABLE 2. Effect of photopeaiodon chemical compositionand histology of parenchyma in the mrmvnary gland of pregnant heifem.

Treatment

Dry matter, @la0 g wet weight Fat, d l 0 0 g wet weight DNA, g DNA. mg/g wet weight RNA, B RNA. mg/g wet weight stromal tissnc, 96 Epithelial cells, 46 Alveolar llrmen, 9b

16L.8D'

8L1d

SE3

P

33.4 11.3 26.1 4.05 18.7 2.87 38.9 26.1 35.0

31.5 9.3 29.8 4.16 21.9 2.96 36.7 26.4 36.9

1.10 1.70 4.04 .120 3.03

.16 .44 .32 .83 38 .35 .35 .79

.a64 559 .69 3.35

29

ISixteen hoan of light and 8 h of darkness. *Eight horn of light and 16 h of darlcness.

%ooled SE. Joamal of Dairy Science Vol. 74, No. 1, 1991

104

"BOLD

ET AL.

TABLE 3. Effect of photopexiod on pregnant heifer growth and carcass and fetal characteristics. Treatment Heifer growth Initial BW, kg Pioat BW, kg Liveweight gain, kg/d carcass characteristics Carcass weight, kg Perired fat, kg S U b c u ~ U sBt, mm Longissimus dorsi area, c m ' Fetal dmacmlm . 'cs Cmwn to rump length, rum Fetal we@& kg

16L:8D1

8L16D2

SE3

P

481 592 .93

525 615 .75

105 75

.02 32 .I 1

282 14.0 3.9 65.9

304 15.0 42 69.9

2.4 .79 34 1.73

978 31.9

989 34.1

14.8 1.49

.06

.914 A54 .is4 2%

.38 .12

lsixteen burs of light and 8 h of darkness. 'Eight hours of light and 16 h of darkness. ~ P SE. ~ M 4probability vdue adjusted for significant covariate (initid BW). ~reatmentmeans were unadjusted.

the correlation coefficient between individual serum prolactin concentration and comsponding temperature at the time of sampling was .05 (P = .18). During the dark period, mean concentration of melatonin in serum and area under the curve were greater than similar measurements made during the light period (Pc.01; Figure 2). Although duration of elevated serum melatonin concentration in the dark period was longer in heifers given 8L:16D, the magnitude of this nocturnal surge was lower than in heifers exposed to 16L:8D (Pc.01). Thus, there was no effect of photoperiod treatment on serum concentration of melatonin throughout the 24-h period (expressed either as total area under the curve over 24 h or as mean daily concentration of melatonin in serum).

exposed to 16L:8D, total area over the 48 h did not differ between photoperiod treatments. Periparturient Surge in Concefitration of Serum Prolactin. All animals exhibited a surge in concentration of prolactin in serum between 72 h before and 48 h after parturition (Figure 4). In comparison with 8L:16D, exposure to 1&8D resulted in a greater peak concentration of serum prolactin and an increased peak amplitude of the prolactin surge (P<.Ool; Table 4). Simi-

I1401 20

Experlment 2

t

Basal Concentrations of Serum Prolactin 2o and Melatonin. Basal prolactin in serum aver160 I97 2 I9 aged 28 and 14 f 3 n g / d (P<.Ol) far heifers DAY OF PREGNANCY exposed to 16L:8D and 8L:16D, respectively. Concentration of melatonin in serum was Figwe 1. Serum prolactin concenfration in pregnant greater (P<.Ol) in dark than in light periods heifers exposed to 16 h of light and 8 h of darkness (U) or (Figure 3). Although duration of the nocturnal 8 h of light and 16 h of darkness (A). Eachpoint repnseats the treatment mean of samples collected from nine heifm rise in melatonin concentration in serum was at XLmhinterVals for6 h. Treatmentswere initiated at 128 longer for heifers under 8 L 16D than for heifers d of pngnancy. Pooled SE was 8.6 ng/ml of s e n m ~ J o d of Dairy Science VoL 74, No. 1, 1991

105

PKOTOPEROD EppEcrs DURING PREGNANCY D A Y O F PREGNANCY 0 160 A 197 0 214

n

Ad?

=

180 120 80 40 0

40

0 8 0

12

24 0

12

24 0

12

24

16

24

TIME O F DAY ( H I

2. srmm melatonin CoDictntratiOn in pregnant heifers exposed to 16 h of light and 8 hof darkmss (A) or 8 hoflight and 16 h of dmkrtess (B) inExperiment 1. The hatched bar represmts the dark period. Pooled SE was 15.7 pg/ml of serum. F1gUl.e

8

16

24

8

TIME OF DAY ( H )

Pigan 3. Sermn melatonin conocntration in pregnant heifem exposed to 16 hof light and 8 h of darkness (A) or 8 hof light and 16 hof darkness (B) i n m e n t 2. The hatched bar repnscnts the dark period. Pooled SE was 15.5

Pgm

Of

-

used as indices of cell numbers and secretory larly, area of senun prolactin for either the activity, respectively. photoperiod failed to afentire surge or for the surge truncated at partu- fect the proportion of p n c h y m a l cells in rition (which eliminated the confounding fac- mammary glands of pregnant heifers. ”he abtors associated with moving the animals 2 h sence of an effect of photoperiod on these after parturition to a second housing site) was variables during pregnancy contrasts with greater for animals exposed to 16L8D compared with 8L:16D. However, photopefiod did not alter duration of the surge or timing of the peak relative to either the start of the surge or to parturition. “here was no effect ( h . 1 ) of photoperiod on either presurge prolactin (27 vs. 30 f 7 ng/ ml of serum), or postsurge prolactin (56 vs. 57 f 7 n g / d of serum). However, postsurge concentration of prolactin was greater than presurge concentration (P<-05). DISCUSSION

Capacity of the mammary gland to synthesize milk is a function of the number and secretory activity of epithelial cells (24). In Experiment 1, total amounts of parmchymal DNA and RNA in the mammary gland were

= L 5

300

I -\Lip

-32 -24 -16 -8 0

8

16 24 32 40 48 56 64

TIME RELATIVE TO PEAK ( H I

Pigure 4. Serum prolactin concentration (mean f SE) dnringtheperiparbDnent . prolactin mge in pregnant heifers exposed to 16 h of light aud 8 h of dadrness (0) or 8 h of light and 16 h of darkness (A). prolactin concentfation was m e r e d relative to peak values. P = Partwition.

Journal of Dairy Science. Vol. 74. No. 1, 1991

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NEWBOLD ET AL.

TABLE 4. Effect of photoppiod on the peripartarient surge of semrn prolactin in pregnant heifers. ~~

16L:8D1

Peak4 ngM Peak amplitude: ngM kea under surge: ng/ml per hour Total surge SM~C tnrncatsd at partnrition Duration of surge, b Timing of peak Internal from start of surge to peak, h I n t e n d fmm peak to parturition, h

8L16D2

467

262

440

244

10,627 7229 68.0

6410 4183

20.7 13.3

SE3 24 24 10%

64.0

757 4.9

14.7 18.0

3.7 2.3

P COOl COOl .04 .03 .84

25 .16

'Sixteen b u r s of light and 8 h of darkness. 'Ei@t hours of light and 16 h of darkness. 3 ~ ~ SE. 1 4

h

u

m prolactin concentration in serum attained during surge.

5swgC peak minus prfmrgc co-don. 6~

under the surge minus presmge concentration.

previous findings (16), in which greater total parenchymal weight and total amount of parenchymal DNA were present in both pre and postpubertal, nonpgnant heifers exposed to 1 C 8 D compared with 8L:16D. Mammary growth is exponential during pregnancy, with most growth occurring in the last trimester of pregnancy (21). In contrast, mammary growth in peripubertal, nonpregnant heifers is much slower. Thus, we speculate that toward the end of pregnancy, mammary cell division may be proceeding at a maximal rate and perhaps cannot be stimulated further. This may explain why 16L8D stimulated mammary growth in prepubertal and postpubertal heifers (16) but not in the pregnant heifers of the present study. Alternatively, photoperiod may be a more potent stimulant of mammary growth in prepubertal and postpubertal, nonpregnant heifers because growth of the mammary gland in the pregnant heifers was approaching maturity. Consistent with this lack of effect of photoperiod on mammary development, photoperiod did not affect serum concentration of progesterone, a hormone that stimulates mammary alveolar development (26). In contrast, 1&8D increased s e m concentration of prolactin, which may be necessary for normal mammary gland development (22). However, the photoperiod-induced increase in concentration of proJ o d of Dairy Science Vol. 74. No. 1, 1991

lactin in serum was not associated with changes in mammary development. In sheep (10) and hamsters (7), duration of the nocturnal rise in serum melatonin concentration, rather than the average concentration of melatonin in serum during dark periods, is the cue for activation of reproductive activity. In our experiment, duration of the nocturnal increase in serum melatonin concentration was greater in pregnant heifers exposed to 8L16D, but t h i s was not associated with reduced mammary growth as might have been expected based on previous results in prepubertal heifers (Sanchez-Barwlo et al., 1991, unpublished da-

ta>.

In studies with nonpregnant heifers, 16L8D stimulated live weight gain compared with 8L: 16D (14, 15). However, in the present study pregnant heifers responded to 1&8D with only a tendency for increased live weight gain. Photoperiod did not affect perirenal fat weight or subcutaneous fat depth of the pregnant heifers. Similarly, melatonin feeding did not affect carcass fat in postpubertal heifers approaching maturity with respect to fat deposition (30). photoperiod did not affect longissimus dorsi area in either the present experiment or in that of Zinn et aL (29). Thus, in pregnant animals approaching maturity with respect to mammary development and body size, photoperiod had no

PHOTOPERIOD EFFJXIS DURING PREGNANCY

107

effect on mammaq or carcass growth and com- concentration, or increased peak amplitude is important in stimulating lactogenesis, perhaps a position. In pevious research, the peak concentration 16LSD photoperiod during the periparhuient of prolactin in serum of peripartmient cattle prolactin surge could stimulate subsequent milk was greater in summer than winter months, but yield In summary, photoperiod had no effect on surge amplitude of prolactin was relatively constaat throughout the seasons (4). In contrast, in mammary gland development during pregnanExperiment 2 of the current study, 16L.8D cy, but, in comparison with 8L16D, a photopeincreased both peak concentration and ampli- riod of 1&8D increased the peak amplitude tude of the penparturient surge of pmlactin. and area of the Periparturient surge in serum Previous reports show that prolactjn returns prolactin. to presurge concentration in serum by 48 h after parturition (1, 8). However, in our experiACKNOWLEDGMENTS ment, concentration of prolactin in setum in the majority of animals had not completely This research was supported in part by the returned to the presurge concentration by this Michigan Agricultural Expexirnent Station and time. The increased postsurge concentration of USDA grants 88-34122-3443 and 87-CRCRprolactin in serum of animals moved to the 1-2302.The authors appreciate the assistance of second site may have been associated with Kelan Moore in developing the prolactin antiincreased ambient temperature, stress, or milk- bodies. The help of Brent Buchanan, Geof3i-e~ ing, factors known to increase concentration of Dahl, T N Hughes, ~ and Tom Forton is grateprolactin in semm (12, 25, 28). Because these fully acknowledged. factors were confounded, their individual contribution to the elevated postsurge conmtraREFERENCES tion of prolactin was impossible to assess. R. U,D. E. Bauman, A. V. Capco, G. T. Two chamctenstics in m u m prolactin found Goodman, and H. A. Tucker. 1981. prolactin regulain the present study diffeml from earlier pubtion of milk secretion and biochemical d i f f d o n lished findings from OUT laboratory. F i t , peak of pp~mmaby epithelial cells in puiparbnient cows. prolactin (467ngfml) was greater than that (194 Endacrinology 10493. PAkas, R. M.,D. E.Bamnan, G.T. GooQnas A V. ng/ml) previously reported (1). Second, esticapnco,a d H. A. Tuckex. 1981. Prolactin regulathm mates of the time of initiation of the prolactin * 'on of "8f""r epithelial of wbe surge in serum also varied, ranging from 24 h . cows. Bndocnmlogy 1W31. cells in parpsrtanent before parturition (8), to the time of parturition 3hsociation of Offkid Analytical chemists. 1965. (l), to 13.3 to 18.0 h before parturition in the Offkid mthods of analysis. loth ed. Assoc. m c . Anal. them.. washhgtox&Dc. present study. These differences could be due 4Chew,B.P.,P. V.Melvcn,RE.Erb, C.L.Zamt, M to diEferences in assays for prolactin, sampling P.D'Amico, and V. P.CollabraUder. 1979. Variables frequency, number and parity of animals used, BsJociatod wilb peripsmrm traits i n dairy Cows. Iv. or ambient temperature. In earlier work, blood seasonal relation.ships amow , pbotoperiod, and blood plasma prolaclh I. Dairy Sci 621394. was collected from only four cows at 12-h intervals (1). and SO peak serum prolactin c ~ n - 5 a J . L . 1 9 7 8 . D t S ~ a n d ~ ~ o f ~ ~ the snimal and medical scicnccs. 1. Iowa state univ. centration was defined less precisely than m the Res&Ames. present study, in which samples were collected 6 Gill, I. L. 1986. Repeated m a m a m t smsitive tests at 4-h intervals on more cows. The practical for qeimmts witb few animals. J. Anim. Sci. 63: 943. significance of photoperiod on the peripar7 Goldman, B. D..J. h4. Damffv, and L. Yogev. 1984. auient surge in serum prolactin depends on its E f f e c t s of timed mlatoniu infusions on rcpmductive subsequent effects on yield and composition of development in the Djmgarhn laamstex (Phdbpw milk. However, there were insufficient numbers slcngorur). Endocrinology 1142071. of animals in our study to measure meaning8Ingalls, W.G., E. M. Convey, and H.D.Hafs. 1973. Bovint ~ a n m LH, GH, and prolactin dariDg late p g fully the lactational response to photoperiodic -cy, partarition and carly lactation. Roc. Soc. Exp. manipulation of the pedparturient surge in se Biol. Mad. 143161. rn Concentration of prolactin. If exposure to 9Kamovsky, M. J. 1965. A formal-glutaraldeincreased prolactin during the surge (i.e., area hyde fixative of high osmolarity for use in electron microscopy. I. Cell Biol. 27:137. of the surge), increased peak serum prolactin

-

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Concentration of bovine prolactin in serum was quantified using a double antibody radioimmunoassay similar to that described previously from our laboratory (11). In the current assay of prolactin, antisera (fitst antibody) to bovine prolactin (NIH-B4) was produced in guinea pigs, and the second antibody was produced in sheep as previously described (25). Radioiodination of bovine prolactin was as previously described (11). The first antibody was diluted 1:5O,OOO in normal guinea pig serum (GIBCO Laboratories, Grand Island, NY), which had been diluted 1:400 in .05 M EDTA phosphate-buffered saline at pH 7.0. One hundred microliters of diluted antibody per tube produced a total binding of 52%. A dilution of 1:50,000 was used in the remainder of the validation. Specificity of the antisera to bovine prolactin was determined by testing the degree of crossreactivity with bovine FSH, bovine LH, bovine somatotropin and bovine thyroid stimulating hormone at a concentration of 500 ng/ tube. Crossreactivity was less than 1% for each hormone. Recoveries fiom 10 replicates of serum supplemented with 2.5 and 5.0 ng bovine prolactin per tube averaged 97.0 f .l%and 93.0 f .l%,respectively. Standard curves, calculated from a third degree polynomial had an R2 ranging from .994 to 9 8 . The dilution response curve for 20, 40,60, 80, and 100 pl serum per tube paralleled the prolactin reference standard curve. The interassay coefficient of variation was 5.7 to 8.4% in samples ranging from 10 to 50 ng/ml, and the intraassay coefficient of variation was 3.9% at 48.5 ng/mL The lower limit of sensitivity was 1.5 ng of prolacWnd of serum.