Dietary Urea for Dairy Cattle. I. Relationship to Luteal function1

Dietary Urea for Dairy Cattle. I. Relationship to Luteal Function 1 H. A. GARVERICK, 2 R. E. ERB, R. D. RANDEL,3 and M. D. ¢UNNINGHA/vt

Department of Animal Sciences, Purdue University Lafayette, Indiana 47907 Abstract

Isonitrogenous complete mixed rations containing concentrates, corn silage, and ground corn cobs, differing only in source of supplemental nitrogen (Group I - - s o y bean meal and Group I I - - u r e a ) , were fed to Holstein-Friesian heifers for a minimum of 60 days. The purpose was to study the effect of dietary urea on luteal function. Jugular blood and corpora lutea were collected on Day 12 after estrus. Average concentrations of progesterone, luteinizing hormone, corticosterone, and cortisol in blood plasma were approximately the same for both groups. Ten corpora lutea from Group I were heavier (P < .005) than the 12 from Group I I (6.4 ~ .6 g versus 4.3 ± .3 g). The corpora tutea from Group I I were softer and more fragile than those from Group I, but there were no discernible histological differences. The contents and concentrations of progesterone and 20fl-hydroxy-pregnene-3-one in nnincubated corpora lutea were not significantly different between treatments. However, corpus luteum tissue from Group I as compared to Group I I synthesized more progesterone (P < .05) during in vitro incubation in blood plasma (2 hr at 37 C) and contained more total progesterone (P < .05) after incubation. Source of jugular plasma substrate, either from Group I or Group I I , had no significant effect on synthesis of progesterone. The relationship between differences in corpus luteum function due to ration and reproductive efficiency was not determined.

Introduction

Urea is used widely as a source of dietary nitrogen in cattle to replace plant proteins. Certain tureen microorganisms anabolize urea nitrogen via ammonia to protein for subsequent digestion and utilization by the cow. However, producers reluctantly incorporate urea into dairy rations because of inconclusive evidence that dietary urea may decrease reproductive efficiency (1, 6, 13, 203 24, 28, 29). Biological activity of luteinizing hormone ( L H ) , the major luteotropie hormone in cattle (11, 16), is destroyed in vitro by substrates containing 6 ~ urea (30). Destruction is 50 to 70% in the presence of 1 ~ urea (30). I t was theorized that breakdown products of urea caused inactivation of L H rather than urea (30). This suggests that an indirect inhibition of luteal function may occur if L H is partially inhibited in vivo in the presence of endogenous urea or endogenous breakdown products of urea. The objective of the present study was to determine if dietary urea was associated with measurable differences in luteal function of dairy heifers. A brief report on p a r t of the data has been published (8). Materials and Methods

Fifteen Holstein-Friesian heifers, 12 to 18 months of age and weighing 270 to 550 kg, were divided into two groups equalized for age and body weight. Group I (seven heifers) was fed soybean meal and Group I I (eight heifers) was fed urea (145 to 240 g / d a y for individuals) as sources of supplemental nitrogen. The two rations were isonitrogenous. Premix concentrate rations were prepared for each group (Table 1) for mixing with the roughage portion. The complete mixed total ration on a dry matter basis contained 22% premix, 56% corn silage, and 22% ground corn cobs. Average ad libitum feed consumption by individuals ranged from 6 to 9 kg of dry matter daily. Approximately 50% of the nitrogen in the total ration for Group I I was from urea. Dietary urea, increased gradually for three weeks, was fed at the experimental level

Received for publication May 17, 1971. 1 Journal Paper 4434, Purdue University Agricultural Experiment Station. 2 Fellow, Allied Chemical Foundation, New York City. Present address: Department of Dairy Husbandry, University of Missouri, Columbia 65201. 3 Present address: United States Department o~ Agriculture Range Livestock Experiment Station, Miles City, Montana 59301. 1669

1670

GARYERICK ET

TABLE 1. Ingredients of the premix concentrate rations. Group Ingredient Soybean meal a Urea b Rolled shelled corn Molasses Sodium chloridec Dicalcium phosphate Vitamins A and D d Total

I

II

(kg)

(kg)

74.40 ........ 15.35 5.00 2.50 2.00 .75

........ 10.94 78.81 5.00 2.50 2.00 .75

100.00

100.00

a 50% Crude protein. b 281% Crude protein equivalent. e Contained trace minerals. d Each kilogram of premix contained 16,875 IU of vitamin A and 3,000 I U of vitamin D. for a minimum of 60 days prior to estrus (Day 0) which started the cycle during which jugular blood and the corpus h t e u m (CL) were collected. Blood plasma was the substrate during incubation of CL tissue to determine if inhibitors of in vitro synthesis of progesterone were present. Equal amounts of blood plasma, collected at 7 A~, were combined from individuals within each group to make two pools of plasma. This was done prior to the day the first CL was excised. The fresh plasma of each pool was divided into aliquots prior to storage at --23 C to provide a control source of frozen plasma each day CL were incubated. A third substrate, the animal's o w n plasma, was collected at 7 A~ on the day of CL removal. A portion of this plasma was stored at 5 C until used for incubation 1 to 4 hr later. The remainder was stored at --23 C until assayed for content of luteinizing hormone, progesterone, corticosterone, and cortisol. During all collections approximately 200 ml of blood were collected by jugular venipuncture into plastic bottles containing heparin. The blood was chilled in ice water and centrifuged at 14,600 × g for 20 rain to isolate the plasma. Twenty-two corpora lutea were removed by midlumbar laparotomy 12 days after estrus. Fifteen were removed from previously unoperated animals and seven (three in Group I and four in Group I I ) were removed during the second estrous cycIe following removal of the first corpus luteum. F r o m rectal palpation it was known that ovulation occurred within JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 11

AL.

two days after estrus. Immediately after removal each CL was chilled in physiological saline, trimmed of excess tissue, weighed and halved. A wedge-shaped sample of the corpus luteum was fixed in Bouin's fluid and stored in 70% ethanol prior to final processing and staining with hemotoxylin and eosin. The remainder of the corpus luteum was minced into pieces less than 1 mm in diameter, transferred to a Buehner funnel (12 cm) over filter p a p e r (Whatman 42) and washed three to five times with physiological saline to develop responsiveness of the tissue to L I t (17). Duplicate samples of approximately 150 mg of luteal tissue, added to 5 ml of the animal's plasma collected before laparotomy, were immediately stored at --23 C until assayed for content of progesterone and 20fl-hydroxy-pregnene-3-one (20fl-ol). I n addition, tissue from 10 of the CL, five each from Groups I and I I , was incubated for 2 hr in a Dubnoff metabolic shaker at 37 C under a stream of 95% 02: 5% CO 2. Approximately 150 mg of the minced tissue from each CL was incubated in duplicate in the presence of 5 ml of plasma from each of the three sources, namely, the animal's fresh plasma and stored (frozen) plasma from Groups I and I I , respectively. A f t e r 2 hr the incubates were stored at --23 C until assayed for content of progesterone and 20fl-ol. LuteM progesterone and 20fl-ol were isolated and quantified by Harms and Malven's (12) procedures. Briefly described, the method included addition of progesterone-4-14C to correct for procedural losses, homogenization, ether extraction, partial purification of the extract by two dimensional thin-layer chromatography, and quantification by gas liquid chromatography. Jugular plasma progesterone, cortisol, and corticosterone were quantified in duplicate by the competitive protein binding technique described by Murphy (18) as modified by Randel et al. (21). Plasma LI-I was assayed in duplicate by double antibody radioimmunoassay (19). Analysis of variance procedures as outlined by Steel and Torrie (27) were used to establish probabilities for significant differences between variables. Results and Discussion

Plasma hormones. Average progesterone, luteinizing hormone, corticosterone, and cortisol in blood plasma were not significantly different between groups, (Table 2) and were similar to other data for Day 12 of the estrous cycle (9).

1671

UI%EA A N D L U T E A L F U N C T I O N

TABL~ 2. Average concentrations of hormones in jugular blood plasma from Groups I and I I . Group Comparison Progesterone Luteinizing hormone b Corticosterone Cortisol

I

II

(ng/ml) 14.4 -+- 2.0 a .46 ±

(ng/ml) 15.1 ± 1.4

.10

.46 ±

2.3 ± .6 3.8 ± 1.1

.12

3.4 ± .9 3.1 ± 1.0

a Standard error. b Nanogram, National Institutes of Health luteinizing hormone, B 6 per milliliter.

Corpora lutea. The CL from Group I averaged 6.4 ± .6 g as compared to 4.3 ± .3 g for Group 1I (P < .005). Only three of the 12 CL from Group I I weighed more than 5 g as compared to 7 of the l 0 CL from Group I. The Group I I average of 4.3 g for Day 12 of the estrous cycle is lower than averages for several experiments summarized by E r b et al. (5). I n comparison, the Group I average is slightly higher (5). Group I I CL were softer, more fragile, and more difficult to express from the ovary because of the lack of firmness as compared to those from Group 1. However, no differences in cellular structure (7) were detectable during histological examination. Moreover, there was no evidence in either group of structural changes indicative of regression as described by Foley and Greenstein (7). The average concentrations of progesterone (nonsignificant) and 20fl-o] (P < .10) were lower in CL from Group I than from Group I I (Table 3). Average content of progesterone was higher (nonsignificant) for Group I because of larger CL as compared to Group I I . The average content of 20fl-ol was nearly the same because the concentration was about 100% higher for Group I I than for Group I. The averages of 192 and 154 #g of progesterone

per corpus luteum in Groups I and I I are comparable to other data for a similar period of the estrous cycle as summarized by Erb et al. (5). Average 20fl-ol was 11 and 159/o of the total progestin as compared to 1 4 ~ reported by Gomes et al. (10). Higher proportions of 20fl-ol have been reported in CL during the late luteal phase (14) and during some stages of pregnancy (4). Such increases in proportion of 20fl-ol to progesterone have been associated with maturity of CL in conjunction with changes in cellular structure (14) which are believed to be regressive (7). Although dietary urea (Group I I ) was associated with decreased CL weight there was little evidence of delayed maturity since these CL contained slightly higher proportions of 20fl-ol to progesterone than controls (Group I ) . Synthesis of progest~ns. There were no significant differences between sources of plasma used as substrate and rate of synthesis of the two progestins (Table 4). Average luteal concentration and content of progesterone and total progestin were significantly (P < .025) higher in the plasma incubates than in unincubated luteal tissue (Table 4). Similarly compared, luteal concentration but not content of 20fl-ol was increased by incubation (P < .025). The average luteal concentrations of progesterone, 20B-ol, and total progestins were nonsignifieantly different between groups before and after incubation (Table 4). The average content of progesterone (P < .01) and total progestin (P < .05) was greater after incubation in luteal tissue from Group I as compared to Group I I (Table 4). As calculated from Table 4, luteal tissue from Group I synthesized more progesterone (P < .05) and total progestin (P < .10). Luteal tissue from Group I synthesized an average of 146 /~g of progesterone p e r CL as compared to 77 , g for Group I I for respective increases of 70 and 48%. Similarly compared, total progestin during incubation increased 75% for Group I and 52% for Group I I .

TABLE 3. Average content of progestins in corpora lutea from Groups I and I I . Group Comparison

Progesterone 20fl-olb

I

II

--(,g)--

--(.g/g)--

--(.g)--

--(.g/g)--

192 - - 27 a 25±10

31 ± 3 3±1

154 ± 14 26± 5

36 ± 2 6±1

a Standard error. b 20fl-hydroxy-pregnene-3-one. JOUENAL O1~ ])AHOY SOIENOE VOL. 54, NO. 11

1672

GARVERICK ET AL.

TABLE 4. Relationship of sources of luteal tissue and plasma to amounts of progestins in luteal tissue following in vitro incubation. Source of blood plasma

None s Unfrozen~ Frozen, Group I Frozen, Group I I Combined incubates None

Group I

Group I I

(t~;/g)

(#g/CL)

3 2 ± 7e 60 ± 19~ 56±10 50± 3 55 ± 7

210± 356 ± 379± 334± 357 ±

5±

2

Unfrozen Frozen, Group I Frozen, Group I1 Combined incubates

10± 11± 9± 10 ±

2 4 2 2

None Unfrozen Frozen, Group I Frozen, Group I I Combined incubates

37± 8 70 ± 21 67 ± :13 59± 5 65 ± 8

(#g/g)

Progesterone 49 40± 3 65~ 58 ± 11 99 60----- 6 49 59± 4 40 59 ± 4

20fl-hydroxy-pregnene-3-one 33± 18 9± 2 6 7 ± 18 11± 1 8 0 ± 40 16± 2 6 2 ± 24 18± 5 69 ± 16 15 ± 2

Total progestin 2 4 3 ± 67 49± 3 423 ± 79 69 ± 11 459 ± 138 76 ± 6 3 9 6 ± 71 77± 8 426 ± 54 74 ± 5

(#g/CL) 160±]1 232 ± 45 242±29 236±24 237 ± 18 37-----

8

45± 4 61± 8 72±22 60 ± 8 197±16 277 ± 45 303 ± 33 308±42 297 ± 22

a Unincubated. b Blood plasma from animal contributing corpus luteum tissue. c Standard error. d Smallest corpus luteum had the highest rate of synthesis (82 tLg/g). General discussion. Two differences in luteal function, associated with including urea in the ration (Group I I ) , were reduced weight of CL (P < .005) at ]2 days after estrus and decreased synthesis of progestins, especially progesterone (P < .05), during in vitro incubation. Inhibition of CL growth as noted 12 days after estrus infers suboptimnm stimnlation from L H during the early phases of the estrous cycle since LH is considered the major luteotropic hormone in cattle (11, 16). Results from incubation of luteal tissue in blood plasma of heifers on the two diets to detect the presence of possible inhibitors were inconclusive. Such inhibitors, if present in blood plasma, probably are not urea or ammonia because these levels are similar when diets containing natural protein or urea at nontoxic levels are compared (28, 29). There was more synthesis (nonsignificant) of progesterone when luteal tissue from Group 1 was incubated in frozen plasma from Group I as compared to incubating the same tissue in unfrozen plasma or in frozen plasma from Group II. Though the amounts synthesized were less in ~'0URNAL OF DAIRY SCIENCE VOL. 54, NO. 11

Group I I as compared to Group I the trends were similar to Group I. Anti-luteinizing hormone reduced corpus luteum weight and content of progesterone when administered to heifers on Days 2 to 6 of the estrous cycle, but concentration of progesterone in the CL was unchanged (26). A similar effect was observed when anti-LH was administered to hysterectomized heifers (2). I f luteal function is inhibited as the result of feeding urea, differences in L H and progesterone in blood plasma should be detectable. However, levels of the two hormones were similar in both groups (Table 2). Failure to detect differences between groups in progesterone in peripheral plasma on Day 12 of the estrous cycle may be inconclusive since differences in rates of secretion, metabolism, and excretion could result in maintenance of similar concentrations of progesterone. Although L H produced large increases in bovine ]uteal synthesis of progesterone in vitro (15), increases in concentration of progesterone in peripheral blood plasma were detected only about 50% of the time when LH was injected in vivo (25).

UREA

AND

LUTEAL

Toxic levels of urea in the diet alter electrocardiogram and r e s p i r a t o r y patterns in ruminants (3, 22) which are not detected when nontoxic levels are fed (23). The daily intake of urea by Group I I a p p a r e n t l y was not stressful since levels of cortisol and corticosterone were similar to Group I. Previous studies on dietary urea for dairy cattle generally have been short term and[ multidirectional. D i e t a r y urea had no adverse effect on estrous cycles (13, 24, 28) or services p e r conception (1, 13) in some experiments and adverse effects in other experiments (6, 20, 28, 29). 01tjen (20) r e p o r t e d that dietary urea which furnished nearly 100% of the dietary nitrogen in purified diets was associated with shortened estrous cycles, increased services p e r conception, longer gestations, increased rates of a b o r t i o n and decreased birthweight of calves. Virtanen (28, 29) has r e p o r t e d increased services p e r conception f o r dairy heifers receiving 99% of their dietary nitrogen f r o m urea and ammonium salts. Including vitamin E in the diet was ineffective f o r i m p r o v i n g conception rates (29). The present study indicates that luteal growth and capacity to synthesize progesterone in vitro may be reduced when 50% of the supplemental nitrogen from urea is substituted f o r p l a n t protein in the diets of heifers for 60 or more days. However, design of this experiment precludes evaluation of effects of the differences on reproductive efficiency. Since the relationships between differences in ]uteal weight and capacity of functional tissue to synthesize p r o gesterone in vivo and reproductive efficiency are unknown, no adverse effects on reproduction due to dietary urea can be ascertained f r o m this study.

Acknowledgments The authors thank Dr. L. E. Reichert, Jr., Emery University, At]anta, Georgia for providing purified bovine lutelnizlng hormone, Dr. G. D. Niswender, University of Michigan, Ann Arbor for providing anti-bovine luteinizing hormone antisera and the Allied Chemical Corporation, New York City for partial financial support.

References (1) Archibald, J. G. 1943. Feeding urea to dairy cows. Massachusetts Agr. Exp. Sta. /~es. Bull., 406. (2) Brunner, M. A., and W. Hansel. 1966. Effects of oxytocin, estradiol, equine L]~ and antibovine LH on luteal function in hysterectomized heifers. Abstr. Federation Prec., 25 : 444.

FUNCTION

1673

(3) Davis, G. K., and ]~. F. Roberts. 1959. Urea toxicity in cattle. Florida Agr. Exp. Sta. Bull., 611. (4) Erb, R. E., V. L. Estergreen, Jr., W. 1~. Gomes, E. D. Fiotka, and O. L. Frost. 1968. Pr0gestin levels in corpora lutea and progesterone in ovarian venous and jugular vein blood of the pregnant bovine. J. Dairy Sci., 51: 317. (5) Erb, R. E., R. D. l~andel, and C. g. Callahan. 1971. Female sex steroid changes during the reproductive cycle. Prec. I X Bien. Symp. Animal Reprod., J. Animal Sci., 32(Suppl. 1 ) : 8 0 . (6) Feoktistov, L. I. 1966. Effect of urea in feed on reproduction in cows. Zivotnovodstvo 8:90 (Nutrition Abstr. Rev., 37: 920). (7) Foley, 1~. C., and J. S. Greenstein. 1958. Cytological changes in the bovine corpus luteum during early pregnancy. Prec. I I I Symp. Reproduction and Infertility. F . X . Gassner, ed. Pergamon Press, London, p. 89. (8) Garverick, H. A., M. D. Cunningham, and R. E. Erb. 1970. Influence of dietary urea on corI)ora lutea. Abstr. J. Animal Sei., 31 : 1033. (9) Garverick, H. A., R. E. Erb, G. D. Niswender, and C. J. Callahan. 1971. Reproductive steroids in the bovine. I I I . Changes during the estrous cycle. J. Animal Sci., 32: 946. (10) Gomes, W. R., V. L. Estergreen, Jr., O. L. Frost, and R. E. Erb. 1963. Progestin levels in jugular and ovarian venous blood, corpora h t e a , and ovaries of the nonpregnant bovine. J. Dairy Sci., 46: 553. (11) Hansel, W. 1966. Studies on the formation and maintenance of the corpus ]uteum, p. 346. In G. E. Lamming and E. C. Amoroso ed, Reproduction in the Female Mammal, Plenium Press, New York City. (12) Harms, P. G., and P. V. Malven. 1969. Modification of bovine luteal function by exogenous oxytocin and progesterone. J. Animal. Sci., 29:25. (13) Lizal, F. 1967. Suitability of large amounts of urea, molasses and straw for rearing heifers. (Cz). Zivoc. Vyr., 12: 357. (Nutrition Abstr. Rev. 38: 264). (14) Mares, S. E., 1~. G. Zimbelman, and L. E. Casida. 1962. Variations in progesterone content of the corpus luteum of the estrual cycle. J. Animal Sci., 21: 266. (15) Mason, N. R., J. M. Marsh, and K. Savard. 1962. An action of gonadotropin in vitro. J. Biol. Chem., 237: 1801. (16) Mason, N. 1~., and K. Savard. 1964. Specificity of gonadotropin stimulation of progesterone synthesis in bovine corpus luteum in vitro. Endocrinology, 74: 664. (17) Moody, E. L., and W. Hansel. 1969. L H stimulated progesterone biosynthesis in JOUR~AL 0F DAII~Y SCIEl~E VOL. ,54, NO. ~I

1674

(18)

(19)

(20)

(21)

(22)

(23)

G A R V E R I C K ET AL.

washed minced luteal tissue preparations. Endocrinology, 84 : 451. Murphy, B. E. P. 1967. Some studies of the protein binding of steroids and their applications to the routine micro and ultramicro measurement of various steroids in body 'fluids by competitive protein binding radioassay. J. Clin. Endocrinol. Metab., 27 : 973. Niswender, G. D., L. E. Reichert, A. R. i~idgley, and A. V. Nalbandov. 1969. Radioimmunoassay of bovine and ovine luteinizing hormone. Endocrinology, 84 : 1166. Oltjen, R. R. 1967. N P N as the primary nitrogen source for cattle. Proc. Cornel] l~utrition Conf. Feed Mfgr. Ithaca, New York, p. 48. Randel, R. D., B. L. Brown, R. E. ]~rb, G. D. Niswender, and C. J. Callahan. 1971. Reproductive steroids in the bovine. II. Comparison of freemartins to fertile heifers. J. Animal Sci., 32: 318. Rash, J. J., M. Muhrer, and R. A. Bloomfield. 1967. Identification of respiratory acidosis during ammonia toxicity. Abstr. J. Animal Sei., 26:1488. Rumsey, T. S., J. Bond, and R. R. Oltjem 1969. Growth and reproductive performance of bulls and heifers fed purified and natural

JOI~N~

O~ D ~ Y

So~o~

~ro~. 54, NO. 11

(24)

(25)

(26)

(27)

(28)

(29)

(30)

diets. II. Effect of diet and urea on electrocardiograph and respiratory patterns. 5. Animal Sci., 28" 659. Rupel, I. W., G. Bohstedt, and E. B. Hart. 1943. The comparative value of urea and linseed meal for milk production. J. Dairy Sei., 26 : 647. Schomberg, D. W., S. P. Coudert, and R. V. Short. 1967. Effects of bovine lutelnizing hormone and human ehorionic gonadotropln oa the bovine corpus luteum in vivo. J. Reprod. Fertil., 14: 277. Snook, R. B., M. A. Brunner, and R. R. Saatman. 1967. Effect of antibovine h t e i n i z i n g hormone in the cyclic heifer. Abstr. J. Dairy Sci., 50: 1000. Steel, R. G. D., and J. tI. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Boo]( Co., Inc., New York City. Virtanen, A. I. 1966. Milk production of cows on protein-free feed. Science, 153: 1603. ¥ i r t a n e n , A. I. 1969. On nitrogen metabolism in milking cows. Federation Proc., 28 : 232. ~isukatol, P., E. T. Bell, J. A. Loralne, and ~. B. Fisher. 1966. The effect of urea on the biological activity of pituitary gonadotropins. J. Endocrinol., 36:15.