EFFECTS OF VARIOUS HORMONE TREATMENTS ON INDUCTION OF LACTATION IN THE EWE l

EFFECTS OF VARIOUS HORMONE TREATMENTS ON INDUCTION OF LACTATION IN THE EWE l H. H. Head 2, C. Delouis 3, M. Terqui 4, G. Kann 3 and J. Djiane 3 University of Florida 2, Gainesville 32611; lnstitut National de la Recbercbe Agronomique 3, 78350 Jouy-en-Josas, France, and Department de Pbysiologie Animale 4, 3 7380 Nouzilly, France Summary In Exp. I and II, 52 of 68 ewes were induced into lactation with twice-daily injections of estradiol-17~ (E2-/~) and progesterone (P4;.5 and 1.25 mg/kg body weight/day) for 7 days. Additional treatments were twice-daily injections (days 18 to 20) of hydrocortisone, growth hormone, thyroxine and thyrotropin releasing hormone alone or in various combinations. In Exp. III, 12 ewes were induced into lactation. In this experiment, all ewes were injected with E2-/3 and hydrocortisone, as previously, but four ewes (III-2) had P4 injections extended to day 20, and four ewes (I11-3) were not injected with P4. Across experiments, lowest milk yields during lactation and the lowest percentage of ewes induced into lactation (58%) occurred when only E 2-~3 and P4 were injected. Inclusion of hydrocortisone injections (50 mg/ day) induced the highest percentage of ewes into lactation (86%, 38 of 44), the highest peak daily yields of milk and the highest total yields during lactation. Including injections of growth hormone, thyroxine or thyrotropin releasing hormone alone or in combinations did not produce better results than injections of E~-/3 and P4 alone. Injections of E2-/~ and hydrocortisone without concurrent injections of P4 were less effective. Intramuscular injections of P4 (10 mg/day) from days 8 to 20 did not inhibit lactogenesis or subsequent lactation. Across all experiments, 76% of multiparous (52/68) and 50% of nulliparous (6/12) ewes produced >100 ml milk/day during their lactation (34 to 95 days). However, yields of milk for ewes that lactated were only 25 to 50% of those from postpartum ewes. The ~Florida Agr. Exp. Sta. Journal Series No. 1770. 2 Dairy Sci. Dept. 3 Laboratoire de Physiologie de la Lactation. 4 Station de Physiologie de la Reproduction. importance of including injections of hydrocortisone in the induction procedure was established, but determination of optimum time to inject and potential importance of other hormones requires additional research. (Key Words: Induced Lactation, Ewes, Estrogen, Progesterone, Milk Yield.) Introduction Lactation has been induced in large and small ruminants by the injection of estrogen and progesterone (Cowie et al., 1952; Fulkerson and McDowell, 1974, 1975a,b; Fulkerson et al., 1975; Smith and Schanbacher, 1973; Collier et al., 1975; Delouis et al., 1978). Yields of milk during induced lactations often were not high; and percentage of animals injected that lactated was variable. Ewes have been induced into lactation by injections of estradiol benzoate and progesterone every 3 days for 60 days (priming phase), followed by 6 days of estradiol benzoate and progesterone injections at lower dosages, or injections of dexamethasone trimethylacetate (Fulkerson and McDowell, 1974). Fulkerson and coworkers (1975a,b, 1976) shortened the priming phase to 30 days. Milking, estradiol benzoate, estradiol benzoate and progesterone, oxytocin, dexamethasone and prostaglandin were used to trigger lactation following the 30-day priming phase. Although results were variable, researchers concluded that appropriate hormonal stimuli following the 30-day priming phase would overcome any inhibitory influences and trigger lactogenesis. Ewes, because of handling ease, lower expense for upkeep and importance in some countries as milk producers, are useful as subjects for the study of mechanisms of mammary gland growth and lactogenesis. Objectives of the present study were to develop a short-term method for inducing lactation in intact ewes based upon high dosages of E2-/~ and Pa, as used previously with cows (Smith and Schan- 706 JOURNAL OF ANIMAL SCIENCE, Vol. 50, No. 4, 1980

INDUCTION OF LACTATION IN EWES 707 bacher, 1973), and to evaluate the effect of including injections of hydrocortisone, growth hormone, thyroxine and thyrotropin releasing hormone on number of ewes induced into lactation and yields of milk. Experimental Procedure Exp. I. Multiparous ewes of the Prealpes du Sud breed were allotted randomly to five groups of four each. All ewes received subcutaneous injections of E2-~ and Pa (.5 and 1.25 mg/kg body weight/day) in the neck starting on the afternoon of day 3 and continuing until the morning of day 10. Steroids were dissolved in ethanol (20 and 50 mg/ml), and one-half the daily dose was given per injection (total of 14 injections), at approximately 0700 hr and 1700 hours. Ewes in group I-1 were given only E2-~ and P4. Ewes in groups I-2 to I-5 received these injections plus twice-daily intramuscular injections of the following hormones on days 18 to 20: I-2, hydrocortisone acetate (H, 25 mg); I-3, growth hormone (GH, 15 mg, 1 IU/ mg); I-4, H (25 mg) and GH (15 mg); and I-5, H (25 mg), GH (15 mg) and thyroxine (T,.5 mg). Ewes were milked by hand beginning on the afternoon of day 21 (19 days after the first E2- ~ and P4 injection), and by machine after day 28. Milk yields were recorded daily during hand milking and then 5 days each week through the remainder of the 34-day lactation. Least-squares analysis of variance was used to obtain quartic regressions (the highest order statistically significant) of milk yield for each group, and orthogonal contrasts compared responses among groups. Exp. I1. Multiparous ewes of the Prealpes du Sud breed were allotted randomly to six groups of eight each. Beginning on day 1, ewes in groups II-1 to II-6 were injected for 7 consecutive days with E2- ~ and P4 (.5 and 1.25 mg/kg body weight/day). Ewes in groups II-2 and 11-6 received the same injection plus intramuscular injections of the following hormones at 0700 and 1700 hr on days 18 to 20: II-2, H (25 rag); II-3, T (.25 mg); 1I-4, H + T; 1I-5, thyrotropin releasing hormone (TRH, 50 jug); and II-6, H + TRH. Starting on day 21, ewes were milked by machine twice daily (0700 and 1600 hr), and milk weights were recorded twice each week throughout the 95-day lactation. Quintic order regressions (the highest order significant) for milk yield and mean daily yields of milk for each group were compared by orthogonal contrasts. Exp. IlL Nulliparous Prealpes du Sud ewes were allotted randomly to three groups of four each. Injections were initiated when ewes were 5 to 6 days postestrus. Ewes in group III-1 were given twice-daily subcutaneous injections of E2-/3 and P4 for 7 consecutive days, and intramuscular injections of H on days 18 to 20. Those in group III-2 received the same injections, plus continued P4 injections (5 mg, IM, twice daily) from days 8 to 20. Group III-3 ewes received subcutaneous injections of E2-/~ (.5 mg/kg body weight/day) only for 7 days, and injections of H on days 18 to 20. Twicedaily milking by machine was initiated on day 21, and milk weights were recorded twice each week throughout a 74-day lactation. Regressions for milk yield were used to compare groups, and orthogonal contrasts to test differences among treatments. Results and Discussion Exp. I. Characteristics of induced lactations for the five groups are given in table i, and regressions of milk yield are depicted in figure 1. The week before initiation of milking, a variable but definite increase in udder size was observed but not quantified. Across groups, nine of 20 ewes (45%) gave no milk the first day of milking. During hand milking yields of milk increased rapidly for ewes in groups I-2, I-4 and I-5, slowly for ewes in group I-3 and least for those in I-1. Mean daily milk yields (ml + SE) for the five groups during the first 5 days of hand milking were 39 -+ 10, 91 20, 93 33, 97 20 and 79 16. Lowest mean daily production during lactation was for ewes in group I-1; two ewes had mean production of less than 25 ml/day. Three ewes in II-3 had mean production of less than 50 ml/day; the fourth produced 511 ml/day. Groups that received injections of H, (I-2, I-4 and I-5) had the greatest number of ewes producing more than 100 ml/day (table 1). Regressions of milk yield for the five treatments differed (P<.01). Orthogonal contrasts showed that the response of ewes in I-1 differed from those of ewes in the other groups (P<.01). Furthermore, the response of 1-4 and I-5 ewes, which received injections of H + GH and H + T, differed from the response of I-3 ewes, (P<.01), which received only GH. Thus, the treatment groups that showed the best response in milk yield had received H. No added benefit resulted when GH + T were included (table 1). Effects of GH + T without H were not evaluated.

708 HEAD ET AL. TABLE 1. LACTATION CHARACTERISTICS OF EWES INDUCED INTO LACTATION WITH ESTRADIOL-17/3 AND PROGESTERONE AND ADDITIONAL HORMONES (EXP. i) Treatment groups a I-1 I-2 I-3 I-4 I-5 -- +H +GH +H, GH +H, GH, T Success b 2/4 4/4 1/4 3/4 4/4 Mean yield (ml) c 75 243 165 179 233 Peak yield (ml) c 96 316 207 240 284 Day of peak c 12 10 34 9 32 34-day total (kg) c 2.5 8.3 5.6 6.1 7.9 aall ewes received estradiol-17/3 and progesterone injections for 7 consecutive days; see experimental methods for description of treatments. bdenoted success when mean yield (X)>IO0 ml/day throughout 34-day lactation (number>lo0 ml per day/ number injected per group). CCalculated from quartic regressions, all ewes included. Apparent differences in time to achieve peak milk yield among treatments were not significant (table 1, figure 1), and total yields of milk during lactation did not differ among groups that received H. Mean daily yields of milk during lactation of ewes (number of ewes in parentheses) which had mean production >200 ml/day were, by groups: I-1 <200 ml (0); I-2, 345 ml (2); I-3,511 ml (1); I-4, 265 ml (2); I-5, 271 ml (3). With the exception of one ewe in I-3, results further support the positive effect of H. Fulkerson and McDowell (1974, 1975a) also demonstrated that exogenous glucocorticoid increased treatment response in ewes and cattle. Mammary glands of ewes not given glucocorticoid developed only slightly or even regressed during milking, whereas ewes given glucocorticoid had mammary glands similar to those of ewes lactating after lambing. 4OO 300 * " " ~ GROUP ~R~,~_ 2 200 --- - - ~ "-- 3 I00 ~ ' ;o ; /o ~ ~o ~,.A~ of ~AC~,O. Figure 1. Regressions of milk yield for ewes induced into lactation (Exp. I). See experimental procedure for treatments. I Injections of additional hormones were given to ewes on days 18 to 20, before the initiation of milking, since this period corresponded to the time when prepartum increases in steroid and protein hormones have been observed in pregnant ewes (Hartmann et al., 1973 ; Stabenfeldt et al., 1972; Thorburn et al., 1972). As observed with cows (Smith and Schanbacher, 1973; Erb et al., 1976; Delouis et al., 1978), ewes (Fulkerson and McDowell, 1974, 1975a,b) and goats (Cowie et al., 1952), estrogen and P4 only partially stimulated mammogenesis and (or) lactogenesis. Perhaps timing and (or) quantities of hormones injected were inappropriate. The milking stimulus and removal of accumulated secretions further provoked mammary gland development (mammogenesis or maturation of cells) but did not stimulate development and function equal to pregnancy. Our results confirm that milking did not provoke lactation in ewes that received only E2- fl and P4 for 7 days (tables 1 and 2). Compared to schemes that required 30- or 60-day initial treatments with estrogen and P4 and various injections of triggering hormones (Fulkerson and McDowell, 1974, 1975a,b; Fulkerson et al., 1976, 1977), our results generally were favorable. Exp. II. A second experiment evaluated the effectiveness of short-term E2- fl and P4 treatment plus several additional hormones in inducing ewes into lactation. In this experiment, TRH was used to provoke Prl secretion (Fell et al., 1973 ; Kann et al., 1973), GH was deleted and one-half the amount of T was injected.

INDUCTION OF LACTATION IN EWES 709 TABLE 2. LACTATION CHARACTERISTICS OF EWES INDUCED INTO LACTATION WITH ESTRADIOL-17jff AND PROGESTERONE AND ADDITIONAL HORMONES (EXP. 1I) Treatment groups a I1-1 1I-2 11-3 II-4 11-5 1I-6 -- +H +T +H, T +TRH +H, TRH Success b 5/8 8/8 6/8 7/8 5/8 7/8 Mean yield (ml) c 174 438 194 340 202 304 Peak yield (ml) c 256 525 266 407 362 321 Day of peak c 60 44 48 44 32 40 95-day total (kg) c 16.6 41.6 18.5 32.3 19.1 28.9 aall ewes received estradiol-17~ and progesterone injections for 7 consecutive days; ods for description of treatments. bdenoted success when mean yield (X)> 100 ml/day throughout the 9S-day lactation total ewes injected for group). CEstimated from quartic regressions, all ewes included. see experimental meth- (ewes>lo0 ml per day/ Hormone combinations evaluated are shown in table 2. Characteristics of the induced lactations and regressions depicting milk yield for the six groups are presented in table 2 and figure 2. Criterion for success was denoted as mean production >100 ml/day through the 95-day lactation period. Although 10 of 48 ewes (-~ 20%) did not attain this level, all were milked through 95 days to evaluate the effect of prolonged milking and to permit comparison among groups. All ewes produced some milk but those that were given E2-3 and P4 only (II-1) had lowest daily mean production during each of the first 3 weeks, and throughout the experiment. Although their production continued to increase during lactation, they also had lowest 600 500 ~ GROUP =, 200 =E I00 0 0 I0 20 50 40 50 60 70 80 90 I00 DAY OF LACTATION Figure 2. Regressions of milk yield for ewes induced into lactation (Exp. 1I). See experimental procedure for treatments. peak daffy production (table 2). Orthogonal contrasts of regressions showed that response of ewes in group II-1 differed (P<.01) from that of ewes in other treatment groups. This finding agreed with results of Exp. I (I-1) and those of Fulkerson and McDowell (1974). Highest milk production occurred in ewes also receiving injections of H (II-2, table 2). All ewes in I1-2 had mean daily production greater than 200 ml/day (range of 219 to 826 ml/day). Mean daily milk production of ewes in II-2, II-4 and II-6 was compared to production of those not injected with H (II-1, II-3 and II-5). Mean daily milk yields (361 vs 190 ml/day) and total lactation milk yields (34.3 vs 18.1 kg) were higher with H (P<.01). No differences were observed among groups not injected with H, although yields of milk were not maintained as well in II-5 (figure 2). However, among groups that received H, regressions of milk yield differed (P<.01). Milk yield regressions for ewes receiving only H (1I-2) were higher than those for ewes in groups II-4 and II-6 (P<.01), and groups II-4 and II-6 differed (P<.10). Efficacy of H in inducing ewes to lactate seems firmly established, whereas effects of additional hormones, such as those administered in Exp. I, were not consistent. Injections of T or TRH without H were less effective and may have antagonized effects of H when given with H. The effect of H was to increase the number of ewes that produced milk at a higher level (>200 ml/day). If ewes that failed to achieve mean daily milk yield of > 200 ml/day

710 HEAD ET AL. are deleted, the range of mean daily milk yields for ewes in the six groups is smaller than when all ewes are considered (table 3), although II-2 is still favored. Exp. III. The effects of prolonged P4 injections (III-2) and injections of E2-~ only (1II-3) on induction of lactation in ewes were evaluated. These treatments were compared to the E2-~ P4- H treatment (III-1). Cubic regressions depicting lactations during the 74 day-period and characteristics of the lactation are pre- "sented in figure 3 and table 4. Mean production >100 ml/day denoted success. Milk yields during lactation for individual ewes in group III-1 ranged from 1.1 to 16.2 kg, with a mean yield of 8.8 kg for the 74 days; two ewes produced more than 100 ml/day. Highest mean daily milk yields, peak yields and total yields were observed in group III-2, which received prolonged injections of P4 (table 4). Maintaining higher concentrations of Pa from days 8 to 20 by continuing P4 injections (III-2) did not inhibit mammogenesis and (or) lactogenesis. Hartmann et al., (1973) concluded that withdrawal of P4 initiated lactogenesis in the ewe. In this study, ewes injected with high doses of Pa (125 mg/day, administered to maintain plasma concentrations between 5 and 10 ng/ml following removal of fetuses by caesarian section on day 144) produced small amounts of milk. Kann et al. (1978) prevented lactogenesis in ewes after caesarian section on day 141 by injecting P4 (75 mg) at the time of surgery and again 8 to 12 hr later. They concluded that failure to initiate secretion resulted from P4 TABLE 3. RANGE AND MEAN DALLY MILK YIELDS OF EWES PRODUCING MORE THAN 200 ML/DAY DURING 95-DAY LACTATION (EXP. li) Milk yield a No. of Group Mean Range ewes b (ml/day) / II-1 II-2 282 438 210-460 219-826 5/8 8/8 1I-3 361 282-494 4/8 II-4 398 251-692 6/8 1I-5 311 215-433 4/8 II-6 348 228-407 5/8 aonly ewes producing > 200 ml/day. bnumber > 200 ml per day/total ewes injected per group. 3OO 200 Is ~,. ~ I ~I00 j ''" " S/" 4 GROUP ~ zb ~ 4'0 ~0 6.0 ;o 8'o ~v OF t.aetation Figure 3. Regressions of milk yield for ewes induced into lactation (Exp. Ill). See experimental procedure for treatments. inhibition of Prl after removal of the fetuses since, in the absence of Pa injections, Prl increased and milk secretion was initiated. Results of the present study and those of Fulkerson et al. (1976), which showed no inhibitory effect of P4, suggest that Pa in blood was not maintained at high (inhibitory) concentrations. Alternatively, the positive effects of estrogens, Prl and glucocorticoids injected or released by milking stimuli may have been sufficient to overcome inhibition by P4. Successful treatments (i.e., estradiol benzoate and progesterone, dexamethasone trimethylacetate, oxytocin and prostaglandin [Fulkerson and McDowell 1974, 1975a,b; Fulkerson et al., 1976, 1977] ) seem to depend at least partially upon the increase in Prl and concentrations of plasma P4. Injecting ergocryptine to inhibit the rise in Prl prevents positive effects of these triggers, and milk yields are reduced (Kann et al., 1978; Fulkerson et al., 1976). Ewes that received E2-/~ only had lowest yields of milk. Only one ewe had mean production > 100 ml/day, but, generally, yields of milk continued to increase throughout the milking phase (figure 3). Cowie et al. (1968) reported that prolonged milking of ovariectomized virgin goats resulted in growth of the mammary gland and lactogenesis. Fulkerson and McDowell (1974) also reported that repeated daily milking of sheep induced into lactation by estradiol benzoate and P4 injections provoked increased mammary secretions. In addition, composition of milk after prolonged milking closely resembled the normal composition for sheep. Nonetheless, prolonged milking following E2-/3 and P4 injections in this and earlier experiments failed to provoke milk yields as great as when H also was injected (figures 1, 2 and 3).

INDUCTION OF LACTATION IN EWES 711 TABLE 4. LACTATION CHARACTERISTICS OF EWES INDUCED INTO LACTATION WITH ESTRADIOL-173 ALONE OR WITH MODIFIED ESTRADIOL-173- PROGESTERONE INJECTIONS (EXP. Ill) Treatment groups a 111-1 11I-2 ili-3 Success b 2/4 3/4 1/4 Mean yield (ml) c 119 180 48 Peak yield (ml) c 189 264 85 Day of peak c 67 62 74 74-day total (kg) c 8.8 13.3 3.6 aewes in III-1, III-2 received estradiol-173 and progesterone injections for 7 consecutive days, and Ill- 2 received additional progesterone (10 mg/day) during days 8 to 20, whereas 1II-3 received estradiol-173 injections only; all ewes were injected with H on days 18 to 20. bdenoted success when mean yield (X)>IO0 ml/ day throughout 74-day lactation (number>lo0 ml per day/total ewes injected per group). CEstimated from cubic regressions, all ewes included. Discussion The present experiments demonstrate that ewes can be induced into lactation with injections of E2-3 and P4 administered for 7 days. Inclusion of H resulted in a higher percentage of ewes producing >100 ml/day throughout lactation, decreased variability in yield of milk among ewes within groups and higher total milk yields. Although lactation characteristics were improved, mean daily milk yields were only 25 to 50% of the yields from postpartum ewes from the main flock. Some ewes had milk yields approaching 75 to 80% of expected yields, but reasons for improvement were not established. In contrast, injections of GH, T and TRH administered immediately before milking, either alone or in several combinations but without H, were not as effective. This observation suggests that endogenous concentrations of these hormones were not limiting or, alternatively, that time chosen to make injections, amounts injected or relative quantities of hormones available for the short time before milking were not correct. The 3 days prior to day of first milking (days 18 to 20) were chosen for injection of additional hormones because prepartum surges in important steroid and protein hormones occur just before parturition (initiation of secretion). However, concentrations of hormones in plasma of ewes (H. H. Head, C. DeLouis, M. Terqui, G. Kann and J. Djiane, unpublisbed data) and cows (Erb et al., 1976; Chakriyarat et al., 1978; Delouis et al., 1978) only partially mimic changes observed near parturition, Perhaps equally important, the relative changes in plasma concentrations of estrogens and Pa prior to milking were different from those prepartum. Increases in concentrations of Prl in plasma of cows when E2-3 and P4 injections were stopped (Erb et al., 1976; Delouis et al., 1978; Chakriyarat et al., 1978) had a marked effect on milk yield (Erb, 1977). Yet TRH injections, which cause short-term increases in Prl in ewes (Kann et al., 1973; Fell et al., 1973), did not improve milk yields or percentage of ewes that produced >100 ml/day (table 2). Similar response to TRH has been reported by D'Amico et al. (1976) and Erb (1977). Amount of Prl released, duration of the increase in plasma after TRH injection or relative concentrations of steroid and (or) other protein hormones in plasma may have been inappropriate. Most mammary gland growth in ewes occurs during pregnancy (78%), with only 2% of total growth occurring during lactation (Anderson, 1975). This contrasts with patterns occurring during induced lactation in ewes (C. Delouis, unpublished data). Delouis found that after 3 weeks of milking total DNA in mammary glands increased (P<.01) to levels above the amount present when milking was initiated on day 21. Cows (Smith and Schanbacher, 1973; Chakriyarat et al., 1978) and ewes (present study, figures 1 and 2, and Fulkerson and McDowell, 1974) continued to increase in milk production longer than during a natural lactation, suggesting continued growth and (or) maturation of epithelial cells. Continued increases in mammary gland size also occurred during milking in goats (Cowie et al., 1968). Glucocorticoids are important for growth of the mammary gland and lactogenesis. Injecting ewes pregnant for more than 100 days with H provoked higher tissue RNA to DNA ratios and milk synthesis but did not stimulate increased levels of DNA (Delouis and Denamur, 1967). In contrast, injections of H given to nulliparous ewes for 7 days following Eu-3 and P4 increased total DNA in the mammary gland on day 21 to levels above those in controls not injected with H (C. Delouis, unpublisbed data). Concurrent increases in plasma concentrations of P4 and

712 HEAD ET AL. glucocorticoids during the induction may be antagonistic to initiation of secretion (Hartmann et al., 1973; Kann et al., 1978), but not to growth of the mammary gland. Delaying injections of H until days 18 to 20, when plasma concentrations of estrogens and P4 are low and concentrations of Prl are increasing (H. H. Head, C. Delouis, M. Terqui, G. Kann and J. Djiane unpublisbed data) might favor initiation of milk secretion, accumulation of milk and relative inhibition of mammary tissue growth. Amount of mammary tissue growth, as well as maturation of epithelial cells, are factors that are related importantly to milk yield. Maximizing both processes will be necessary to improve the induction procedures. The ewe seems to be a useful model with which to study induction of lactation as well as to gain a clearer understanding of hormonal and biochemical events associated with growth of mammary tissue and lactogenesis. The current study and those of Erb and coworkers (Erb et al., 1976; Erb, 1977) and Fulkerson and coworkers (Fulkerson and McDowell 1974, 1975a, b; Fulkerson et al., 1976, 1977) provide a data base from which further studies can be developed. Literature Cited Anderson, R. R. 1975. Mammary gland growth in sheep. J. Anim. Sci. 41:118. Chakriyarat, S., H. H. Head, W. W. Thatcher, F. C. Neal and C. J. Wilcox. 1978. Induction of lactation: Lactational, physiological and hormonal responses in the bovine. J. Dairy Sci. 61:1715. Collier, R. J., D. E. Bauman and R. L. Hays. 1975. Milk production and reproductive performance of cows hormonally induced into lactation. J. Dairy Sci. 58:1524. Cowie, A. T., S. J. Folley, F. H. Malpress and K. C. Richards. 1952. Studies on the hormonal induction of mammary growth and lactation in goats. J. Endocrinol. 8:64. Cowie, A. T., G. S. Knaggs, J. S. Tindal and H. Turvey. 1968. The milking stimulus and mammary growth in the goat. J. Endocrinol. 43:651. 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