Effect of/3-Carotene and Vitamin A on Progesterone Production by Bovine Luteal Cells 1

R. L. GRAVES-HOAGLAND 2 , T. A. HOAGLAND, and C. O. WOODY Department of Animal Science

University of Connecticut Storrs 06268

ABSTRACT

The relationship between con- centrations of plasma vitamin A and c-carotene and corpora lutea was studied using 52 Holstein cows. Bovine lutein- izing hormone was added to incubation tubes in doses of 0, 10, or 100 ng/ml. Re- gression of progesterone secretion by luteal cells in vitro on plasma/3-carotene was positive and significant for cor- pora lutea collected during the winter months when plasma/3-carotene was low. The two were unrelated during the summer months when /3-carotene was higher. Similar regressions for in vitro progesterone product ion and vitamin A were not significant in either season. These results suggest that in vivo /3- carotene status is related to bovine lu- teal function in vitro.

I N T R O D U C T I O N

Vitamin A functions in epithelial cell in- tegrity, vision, and skeletal development, and its role in reproduct ion is well-recognized (9). In a series of studies reviewed by Lotthammer (12), evidence was presented for a specific effect of /3-carotene on reproductive perfor- mance of cattle, other than as a precursor for vitamin A. The corpus luteum was suggested as a possible site of action since the bovine corpus luteum contains a high concentrat ion of /3- carotene (6). Lot thammer (12) reported that corpora lutea (CL) of heifers developed more slowly and were smaller, and cows had lower

Received July 1, 1987. Accepted September 30, 1987. ~Scientific Contribution Number 1194, Storrs

Agricultural Experiment Station, University of Con- necticut, Storrs.

2Present address: The American Red Cross, 209 Farmington Avenue, Farmington 06032.

serum progesterone (P4) when dietary /3- carotene was deficient. Several investigators (3, 5, 7, 8, 13) have since failed to demonstrate a positive effect of /3-carotene, ye t other inves- tigators have confirmed a benefit of/3-carotene on ferti l i ty of cattle (1, 14, 21), on P4 pro- duction st imulated by human-chorionic go- nadotropin (4) and on postpar tum involution (17). Most recently, a positive effect of adding /3-carotene or vitamin A to the culture medium on in vitro P4 product ion by porcine luteal cells was demonstrated (23).

The present s tudy was designed to inves- tigate the effects of /3-carotene and vitamin A on bovine luteal cell P4 production. The re- lationship of in vivo plasma /3-carotene and vitamin A with luteal P4 product ion in vitro was examined.

M A T E R I A L S A N D M E T H O D S

Midcycle CL were obtained at a local slaugh- terhouse from nonpregnant Holstein cows. Dispersion and incubation of luteal cells were performed according to the method of Sim- mons et al. (20) with slight modifications. Briefly, luteal tissue was dispersed with col- lagenase (EC 3.4.24.3), and 400,000 viable ceils, as determined by trypan blue exclusion, were suspended in 1 ml of Ham's F12 medium (Flow Laboratories, McLean, VA) and in- cubated for 1 h in a Dubnoff Metabolic Shaking Incubator (Precision Scientific Co., Chicago, IL) at 37°C and 100 rpm. Bovine luteinizing hormone (bLH; NIAMDD-bLH-4) was added to incubation tubes in doses of 0, 10, or 100 ng per tube. The cells were removed by centri- fugation (800 x g for 10 min), and the medium was assayed for P4 by radioimmunoassay according to the method of Hoagland and Barnes (10). Intraassay and interassay co- efficients of variation for the P4 radioim- munoassays were 10.3 and 5.1%, respectively. Preincubation tubes had no bLH added to them

1988 J Dairy Sci 71:1058-1062 1058

EFFECT OF ~CAROTENE ON LUTEAL CELLS 1059

and were not incubated, but they were cen- trifuged as described within 5 min after the addition of cells.

Blood samples were obtained from cows from which CL were obtained to be incubated. A total of 32 CL were obtained during the winters (December through February) of 1985 and 1986 and an additional 20 CL were ob- tained during the summers (June through August) of the same years. Plasma concen- trations of/3-carotene and vitamin A equivalents were determined according to Kimble (11). An Evelyn colorimeter (Rubicon Co., Philadelphia, PA) was used for direct colorimetric deter- mination of /3-carotene equivalents, and the Carr-Price reaction followed by colorimetry was used for the determination of vitamin A equiv- alents. Analysis of covariance was performed to relate P4 production in vitro to plasma /3- carotene and vitamin k . Percent P4 response during incubation was used as the dependent variable, whereas the independent variables used were season of the year as a main effect, and plasma /3-carotene and vitamin A as co- variates. Percent P4 response was defined as the percent change in the medium due to treatment (incubation with 10 or 100 ng bLH) relative to the initial (0 ng bLH) P4 level. Unstimulated percent change in P4 was the difference be- tween preincubation tubes and those incubated with 0 ng bLH. Separate analyses were per- formed for 0, 10, and 100 ng bLH.

R ESU LTS

There was an overall increase (P<.01) in P4 production due to bLH for the 52 CL Pro- gesterone concentration was higher when luteal cells were treated with 10 ng (P<.05) or 100 ng (P<.05) bLH than when 0 ng bLH was added to the incubation tubes. However, there was no difference between tubes that had 10 and 100 ng bLH added (Table 1).

Mean plasma concentrations of /3-carotene and vitamin A equivalents are shown in Table 2. During the summer, mean plasma concentration of vitamin A was nearly 1.5 times higher (P<.01) than during the winter. Although mean plasma concentration of /3-carotene was 1.2 times higher during the summer than during the winter, the difference was not significant (P> .1).

Analyses of covariance relating P4 pro- duction to plasma/3-carotene and vitamin A are

TABLE 1. M e a n (-+SE) progesterone (P4) concentration (ng/h per 400,000 cells) for preincu- bation tubes and those incubated for 1 h with bovine luteinizing hormone (bLH).

Treatment P4 n

( n g ) ~ ,X SE

Preincubation 46.5 a 4.7 52 Incubated with:

0 ng bLH 51.7a 5.0 52 10 ng bLH 64.6 b 6.8 51 100 ng bLH 70.4 b 7.3 52

a'bMeans with different (P<.05).

1MSE = 275.4 (df= 86).

superscripts differ

shown in Table 3. Concentration of P4 in the absence of LH was affected by plasma /3- carotene (P<.05) as well as by the interaction between season and plasma/~-carotene (P<.02). For the increment in P4 production due to stimulation with 10 ng bLH, there was an interaction between season and plasma /3- carotene (P<.03), and season tended (P<.07) to be significant. When 100 ng bLH were added to luteal cell incubation tubes, both plasma /3-carotene (P<.04) and the interaction between season and plasma/3-carotene (P<.04) affected percent change in P4 concentration of the medium.

To examine further the relationship between Pa production by luteal cells in vitro and

TABLE 2. Mean (-+SE) plasma equivalents of /3- carotene and vitamin A of cattle during the winter (December to February) and summer (June to Aug- ust).

Season No. cows /3kCarotene Vitamin A

- - (mg/dl) - - SE X SE

Winter 32 303 35 22.0 a 1.4 Summer 20 378 44 32.1 b 1.7 Overall 52 332 27 25.9 1.3

a,b Means within the same column with different superscripts differ (P<.O1).

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1060 GRAVES-HOAGLAND ET AL.

TABLE 3, Analysis of covariance of percent change in progesterone during incubation with season as a main effect and plasma ~-carotene (PLB) and vitamin A (PLA) as covariates.

Source of Sums of squares variation df 0 ng bLH I 10 ng bLH 100 ng bLH

Season 1 1809.8 5068.1 b 7669.1 PLB 1 3651.1 a 3716.7 14,064.5 a PLA 1 2325.3 589.5 262.4 Season X PLB 1 5220.2 a 8121.7 a 14,094.0 a Season X PLA 1 .02 564.6 374.4

Error 46 33,845.5 64,902.2 132,291.1

ap<.05. bp<.07.

t Bovine luteinizing hormone.

p lasma /3-carotene, p e r c e n t change in P4 was regressed o n p lasma /~-carotene and v i t amin A, separa te ly , du r ing the w i n t e r and s u m m e r (Table 4). Each measure of in vi t ro P4 pro- d u c t i o n was pos i t ive ly re la ted ( P < . 0 1 ) to t h e p lasma c o n c e n t r a t i o n of /3-carotene dur ing t he win ter . This was n o t t r ue for p lasma vi- t a m i n A dur ing t he w i n t e r ( P > . I ) or for e i the r m e t a b o l i t e du r ing the s u m m e r (P<. 1) excep t at 0 ng bLH, w h e r e the re was a t r e n d ( P < . 0 6 ) for v i t amin A to be negat ive ly re la ted to P4 pro- d u c t i o n .

DISCUSSION

Mean p lasma c o n c e n t r a t i o n o f /3 -ca ro tene for cows in the p re sen t e x p e r i m e n t is in close

ag reemen t w i t h the es t imates o f Marcek et al. (13) using HPLC. Fu r the r , m e a n p lasma con- c e n t r a t i o n o f v i t amin A is s l ightly h igher than , a l t hough w i t h i n the range of, values repor ted b y Chew et al. (6), w h o measu red to ta l v i t amin A af te r s epa ra t i on w i t h TLC. P lasma ~fl-carotene and v i t amin A c o n c e n t r a t i o n s were highly var iable a m o n g animals used in the p resen t e x p e r i m e n t (CV = 60% and 36% for t3-carotene and v i t amin A, respect ively) , w h i c h is in agree- m e n t w i t h o the r s (6) w h o sampled s laughter- house cows. This m a y be due to the varied n u t r i t i o n a l b a c k g r o u n d s of the animals. Season- al d i f fe rences in p lasma ca ro t eno id s and v i t amin A o f ca t t le have b e e n r epo r t ed (15, 18).

Regress ion analyses ind ica te a re la t ionship b e t w e e n p l a sma f l-carotene and in vi t ro P4

TABLE 4. Regression coefficients (b) and probability values determined when percent change in progesterone during incubation of luteal cells was regressed on plasma levels of j3-carotene and vitamin A during two seasons.

Winter Summer Plasma

bLH 1 metabolite b P b P

(ng) 0 ~Carotene .10 <.01 - .009

Vitamin A --.94 >.2 --.94 10 fl-Carotene .12 <.01 --.02

Vitamin A .94 >.2 .01 100 p-Carotene .18 <.01 --.0001

Vitamin A .06 >.9 --.69

>.5 <.06 >.6 >.9 >.9 >.6

1 Bovine luteinizing hormone.

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EFFECT OF ~CAROTENE ON LUTEAL CELLS 1061

p r o d u c t i o n by lu tea l cells. P lasma /3-carotene m a y in f luence lu tea l f u n c t i o n w h e n / 3 - c a r o t e n e a n d / o r v i t a m i n A in the d ie t are low, and poss ib ly l imi t ing; i.e., dur ing winter . O t h e r inves t igators (1) have also s h o w n a seasonal in f luence o n t he e f fec t o f /3 -ca ro t ene o n bov ine r e p r o d u c t i o n . Cows s u p p l e m e n t e d wi th /3- ca ro tene had a h igher c o n c e p t i o n rate, b u t th is was observed o n l y in those an imals i n s e m i n a t e d dur ing t h e winter . T e m p e r a t u r e and p h o t o - per iod vary seasonal ly and have b e e n s h o w n to in f luence mi lk y ie ld (16) and fe r t i l i ty (2, 22 ) as well as p la sma c o n c e n t r a t i o n of several h o r m o n e s (15) in Hols te in cows.

This s t u d y does n o t address t he ques t i on o f an ind i r ec t e f fec t o f / 3 - c a r o t e n e caused b y its convers ion to v i t amin A. I t is in te res t ing to cons ider t he absence of posi t ive r e l a t ionsh ips be tween /~ -ca ro t ene or v i t am i n A and in vi t ro P4 p r o d u c t i o n dur ing t h e s u m m e r and a poss ib le negat ive e f fec t o f v i t amin A at t h a t t ime, in l ight o f t h e r e p o r t by R o s e n b e r g et al. (19) of lower P4 se rum c o n c e n t r a t i o n and c o n c e p t i o n ra te of p o s t p a r t u m da i ry cows dur ing t he summer . More research is n e e d e d to inves t iga te t he poss ib l i ty o f seasonal e f fec ts o f /~ -ca ro tene and v i t amin A o n bov ine r e p r o d u c t i o n .

In conc lus ion , the da t a suggest t h a t a pos- i t ive re la t ionsh ip exists b e t w e e n in vi t ro b o v i n e lu tea l cell P4 p r o d u c t i o n a n d p l a s m a / ~ c a r o t e n e dur ing t he w i n t e r w h e n p l a sma ~-caro tene and v i t amin A are decreased. However , dur ing t he s u m m e r w h e n p lasma v i t am i n A and ~-caro tene are e levated, t h e posi t ive r e l a t ionsh ip is los t and m a y b e c o m e negat ive for v i t a m i n A.

ACKNOW LEDGME N"IS

The a u t h o r s acknowledge t he gif t o f LH (NIAMDD-bLH-4) f r o m t he p i t u i t a r y h o r m o n e d i s t r i b u t i o n program, Na t iona l In s t i t u t e s o f Ar- thr i t is , Me tabo l i sm, and Digestive Diseases. G r a t i t u d e is expressed to R. E. Butcher , West Virginia Univers i ty , for the gif t o f an t i sera to p roges te rone . The a u t h o r s wou ld also l ike to t h a n k H o f f m a n - L a R o c h e , Inc. (Nut ley, N J) and Agway Inc. (Syracuse, NY) fo r par t ia l s u p p o r t o f this p ro jec t . We also t h a n k Mary A. Bub for t yp ing th is manusc r ip t .

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