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Growth response, endocrine profiles and reproductive performance of fine-wool ewe lambs treated with ovine prolactin before breeding
THERIOGENOLOGY GROWTH RESPONSE, ENDOCRINE PROFILES AND REPRODUCTIVE PERFORMANCE OF FINE-WOOL EWE LAMBS TREATED WITH OVINE PROLACTIN BEFORE BREEDING R. A. Spoon and D. M. Hallford Department of Animal and Range Sciences College of Agriculture and Home Economics New Mexico State University Las Cruces, NM 88003-0009 Received for publication: November 3, 1988 Accepted: ApriZ 25, 1989
ABSTRACT Twenty-four 6-mo-old ewe lambs received one of two ovine prolactin (oPRL) treatments 28 d before fall breeding. Beginning on the first day of treatment (Day 0), 12 lambs received a subcutaneous injection (12 ml) of a carrier vehicle (0 mg oPRL) on alternate days for 28 d while 12 lambs received injections containing 5 mg oPRL. On Days 0 and 28, jugular blood was collected from six lambs in each group before treatment and at 30-min intervals for 6 h thereafter. Neither feed intake, efficiency of gain nor animal weights differed (P > 0.20) between groups. One hour after treatment on Day 0, ewe lambs receiving 5 mg oPRL had greater (P < 0.10) serum PRL levels than did controls (121.9 and 61.5 + 24.7 ng/ml, respectively). Differences in serum PRL persisted throughout remaining sampling intervals on both Days 0 and 28. Serum samples obtained on alternate days during the 28-d treatment period revealed no differences (P > 0.20) in PRL concentrations between control (48.3 + 5.3 ng/ml) and oPRL-treated (55.7 + 5.3 ng/ml) ewes. Neither serum-insulin nor growth hormone respondyd (P > 0.05) to exogenous oPRL on either Day 0 or 28. No difference (P > 0.30) in percentage of ewe lambs cycling during treatment or breeding was detected between groups. Subsequent lambing percentages were similar (P > 0.30), with 36.4% of control and 25.0% of oPRL-treated ewes producing offspring. Administering 5 mg oPRL on alternate days for 28 d before breeding did not enhance growth and(or) reproductive performance in virgin ewe lambs. Key words:
sheep, prolactin, reproduction, growth hormone, insulin
Acknowledgments: Appreciation is expressed to D. Holcombe, W. Hoefler, R. Oyler and R. Reynolds. We also thank those who supplied assay materials: National Hormone and Pituitary Program (Univ. of Maryland School of Medicine), Dr. A. F. Parlow (Pituitary Hormones and Antisera Center, Harbor/UCLA Lilly Laboratories Research Medical Center, Torrance, CA), (Indianapolis, IN; oInsulin, lot 615-1112B-108-I) and Dr. G. D. Niswender (Department of Physiology and Biophysics, Colorado State yniversity, Fort Collins, CO; progesterone-11 BSA antiserum 1337). Journal Article 1425 of the New Mexico Agricultural Experiment Station. Reprint requests and correspondence to D. M. Hallford.
JULY 1989 VOL. 32 NO. 1
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THERIOGENOLOGY INTRODUCTION Improving reproductive efficiency is a major goal of sheep producers. Although not commonly practiced, breeding ewe lambs is one method that may increase lifetime productivity and longevity of ewes (1, 2). However, reproductive performance in ewe Iambs is generally poor compared with mature ewes because of failure to attain puberty (2), shorter duration of estrus (3), lower lambing rates (4) and higher incidences of early embryonic mortality (5) and general reproductive failures (6). Body size and age appear to be critical factors influencing the onset of puberty in ewe lambs (2, 7). Lambs growing at faster rates generally exhibit earlier estrus and are more likely to conceive at younger ages (4, 8). Methods of accelerating growth rates that do not limit reproduction might, therefore, benefit overall reproductive performance of the ewe lamb. Recent evidence indicates that prolactin (PRL) may be involved in the regulation of overall body growth and nutrient partitioning (9). Research suggests PRL is an anabolic hormone in ruminants (10 - 12) and numerous treatments which increase growth rates also elevate serum PRL (13). Additionally, interactions between PRL and growth have been suggested by studies involving photoperiodic changes. Beneficial gains associated with various photoperiod regimens have been reported in rams (11, 14) and wethers (15) in the presence of elevated PRL concentrations. Thus, distinct roles for PRL in growth have yet to be defined in domestic animals. This study examined effects of exogenous ovine PRL on growth responses, endocrine profiles and reproductive performance of fine-wool ewe lambs.
MATERIALS AND METHODS Thirty-three days before the fall breeding (5 d before initiating treatments), 24 fine-wool (Debouillet x Rambouillet) ewe lambs (6 mo old) were randomly allotted to one of two treatments (12 lambs/group). Lambs in each group were placed in one of four 3.1 x 9.1 m pens (eight pens total, three lambs/pen) to allow determination of feed intake and efficiency of weight gain during a 28-d study. Throughout the study, lambs were allowed ad libitum access to a pelleted alfalfa diet, water, salt, mineral and shade. Average diet composition (dry matter basis) was 93.0% dry matter, 15.6% crude protein, 9.1% ash, 2.3% ether extract, 1.2% calcium and 0.3% phosphorus. Weights were obtained before treatment and at 10-d intervals thereafter, at the end of breeding and near the time of lambing. Beginning on Day 0 (first day of treatment), ewe lambs received subcutaneous injections containing either 0 or 5 mg oPRL (NIDDK-oPRL-17) on alternate days for 28 d. Ovine PRL was solubilized in 0.9% saline and adjusted to pH 9.6 by addition of 0.025 M NaHC03 and 0.025 M Na2C0 (16), such that the final volume (12 ml) contained either 0 or 5 mg OPaL. Jugular blood samples were collected by venipuncture from all ewes on alternate days (beginning on Day 0) throughout treatment and every third day (beginning on Day 29) during breeding to determine the onset of estrual activity. On Days 0 and 28 (after a 12-h fast and another
46
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THERIOGENOLOGY feeding for 1 h), jugular blood samples were collected from six ewes in each treatment group before treatment and at 30-min intervals for 6 h thereafter to examine acute and chronic effects of oPRL on serum hormone profiles. Blood was allowed to clot at room temperature for 45 min, after which serum was separated by centrifugation (2,300 x g, 4"C, 15 min) and stored (-ZO'C) until serum hormones could be quantified by radioimmunoassay. Serum progesterone (17), growth hormone (18) and insulin (19) were determined by previously validated procedures. The PRL assay utilized rabbit anti-oPRL (NIDDK-anti-oPRL-1), the specificity of which has been previously reported (Technical Report 109, Pituitary Hormones and Antisera Center, Harbor UCLA Medical Center, Torrance, CA). Radioiodination of highly purified oPRL (NIDDK-oPRL-I-2) was accomplished by the chloramine odium ?I!? metabisulfite method described by Niswender et al. (20) except I was used as the tracer. Standards were prepared by making appropriate dilutions of oPRL (NIDDK-oPRL-17) in 0.01 M phosphate buffered saline containing 1% bovine serum albumin (PBS + 1% BSA, pH 7.0). Anti-rabbit gamma globulin was raised in sheep (second antibody) using procedures described by Swanson et al. (21) and was suspended at a working dilution (1:lO) in 0.01 M PBS containing 0.05 M ethylenediaminetetraacetic acid (PBS-EDTA, pH 7.0). The assay was accomplished by pipetting standard or serum (usually 0.025 to 0.04 ml) fnto 12 x 75-m disposable plastic culture tubes and then normalizing all tubes to 0.5 ml with PBS + 1% BSA. Rabbit anti-oPRL (0.2 ml of a l:lOO,OOO dilution in PBS-EDTA + 1:400 normal153bbit serum, pH 7.0) was added to each tube followed by addition of I-oPRL (0.1 ml PBS + 1% BSA containing approximately 25,000 cpm). Tubes were vortexed briefly and incubated overnight at 4'C, followed by addition of 0.2 ml of second antibody and a second overnight incubation (4°C). Following final incubation (third day), 1.0 ml cold (4°C) 0.01 M PBS was added and tubes were centrifuged (2,300 x g, 4°C) for 15 min. After decanting the supernate, radioactivity in the bound fraction was determined and hormone concentrations were calculated. Under these assay condfs3ons, a l:lOO,OOO dilution of anti-oPRL routinely bound 51% of added I-oPRL, of which 3.8% was nonspecific binding. Addition of 1.0 ng oPRL to the resulted in approximately a 56% reduction in the amount of BfSay I-oPRL bound to the primary antibody. When 25 ng oPRL were added to ovine serum containing 7.5 nglml, 33.7 + 0.5 ng/ml (mean c SEM) were recovered (104%). Within- (n = 12) and between- (n = 3) assay coefficients of variation were 9.7 and 10.8X, respectively. Following the 28-d treatment period, ewe lambs were placed in a single pen (4.3 x 15.2 m) and joined with fertile Debouillet rams for 34 d. One control ewe became ill during breeding and her data were subsequently eliminated, with the exception of feed intake and efficiency of weight gain, which were determined on a pen basis. Feed intake and efficiency of weight gain were evaluated by analysis of variance for completely random designs (22). Animal weights and serum hormone profiles were subjected to split-plot analysis of variance (23) with effects of oPRL treatment included in the main plot and tested using animal within treatment as the error term. Categorical data (cyclicity and reproductive performance) were
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THERIOGENOLOGY analyzed using Chi-square procedures (24). Analyses of variance and categorical analyses were computed using the General Linear Models and Catmod Procedures, respectively, of the Statistical Analysis System (25).
RESULTS AND DISCUSSION Feed Intake and Ewe Weights Total feed intake (pen basis) during the 28-d treatment period was similar (P > 0.20) between groups, with ewe lambs receiving 5 mg oPRL consuming (mean + SEM) 161.9 + 8.3 kg compared with 153.3 + 8.3 kg for control animals, Likewise, ewe weights were comparable-(P > 0.10) throughout the 28 d of treatment with oPRL-treated ewes weighing 47.9 + 1.1 kg on the final day of treatment (Day 28) compared with 47.5 + 1.i kg for the controls (Table 1). Furthermore, when total feed intak; and total weight gain (pen basis) were examined in terms of efficiency of gain, no differences were detected (P > 0.10) between groups (10.4 and 9.4 + 0.4 kg feed/kg gain for ewe lambs receiving 0 and 5 mg oPRL, respectively). Thus, administering 5 mg oPRL to ewe lambs on alternate days for 28 d did not influence intake, gain or efficiency of gain. Likewise, Eisemann et al. (15) reported that daily administration of exogenous oPRL for 9 wk did not enhance total gain or feed intake in wether lambs maintained in 8 h light:16 h dark compared with controls kept under similar conditions. However, active immunization against PRL (26) or inhibition of PRL secretion with 2-Br-a-ergocryptine (15) in rams and wethers resulted in decreased body weight gains and feed intake. Ewe weights were also obtained at regular intervals following the 28-d treatment period. Weights were similar (P > 0.10) between control and oPRL-treated ewe lambs at the end of breeding (50.1 and 51.8 + 1.2 kg, respectively) as well as near the time of parturition (55.3 and 57.6 2 1.2 kg, respectively). Table I.
Body weights (kg) of ewe lambs Eeceiving ovine prolactin (oPRL) for 28 d before breeding
Item No. of animals
oPRL, mglalternate d 0 5 11
12
41.7 43.5 45.7 47.5 44.6
42.0 43.2 46.0 47.9 44.8
SEMC
Time after initiating treatment (days) 0 9 19 28 Overall meanb
1.0 1.1 1.1 1.1 1.0
*Split-plot analysis of variance revealed no oPRL by day interaction b(P > 0.50); therefore overall means were tested. Row values do not differ (P > 0.10). 'Based on 11 ewe lambs/treatment.
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THERIOGENOLOGY Serum Prolactin In jugular samples obtained immediately before treatment and at 30-min intervals for 6 h after treatment on Days 0 and 28, split-plot analyses of variance revealed oPRL by sampling time interactions (P < 0.10); therefore, effects of exogenous oPRL were examined within sampling hour on each respective day (Table 2). Also, because PRL values varied widely, ranks rather than actual hormone values were analyzed on both days. Serum PRL did not differ (P > 0.10) before and 30 min after treatment on either Day 0 or 28 in control and oPRL-treated ewe lambs. However, by 1 h after treatment on Day 0, ewe lambs receiving 5 mg oPRL had greater (P < 0.10) serum PRL values than did controls (121.9 and 61.5 + 24.7 ngfml, respectively). Throughout the remaining sampling intervals, oPRL-treated ewe lambs generally maintained two- to three-fold higher PRL concentrations than those observed for controls. Similar responses were detected on Day 28. Within 1 h after treatment, oPRL-treated ewe lambs had higher
Table 2.
Serum prolactin (ngfml) in ewe lambs before and for six hours after ovine prolactin (oPRL) administration on days 0 and 28 during a 28-day treatment perioda
Time (hours)
0
5
,oPRL, mgialternate day k Day 28 SEM' 0 5
SEM'
Before treatment 0
90.7h
94.gh
31.1
39.zh
34.Zh
10.6
After treatment 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
87.7h 61.5d 63.5d 56.1d 70.4h 45.1f 61.3h 49.4d 47*4f 58.0f 50.Zf 46.7d
131.0h 121.ge 118.1e 100.6e 115.6h 84.4g 97.gh 86.5e 109.11 151.48 156.2' 125.ge
23.0 24.7 19.8 17.4 23.4 12.4 15.1 13.3 26.4 39.9 39.5 31.3
65.gh 45.7f 62.5d 41.gf 55.1f 31.Zf 38.Zf 24.1f 52.gf 39.6f 28.Zf 37.2f
115.gh 98.Zg 93.ze 103.48 128.3' 106.3' l08.4g 71.6g 116.4g 104.5g 94.3g 105.58
39.4 25.9 11.9 17.8 29.9 17.7 12.5 6.5 22.2 9.6 12.2 13.4
"Five ewes (0 mg oPRL) and six ewes (5 mg oPRL); sampled at 30-min intervals for 6 h after treatment. Treatments were administered b(~.~.) on alternate days for 28 d before breeding. Ovine prolactin by sampling hour interaction was detected (P < 0.10); therefore, treatment effects were examined within hour. Because PRL values varied widely, ranks were analyzed within =time of sampling. Analyses were conducted within sampling day. d ,Based on 5 ewe lambs/treatment. ' Row values within day differ (P < 0.10). f'gRow values within day differ (P < 0.05) hRow values within day do not differ (P ; 0.10).
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THERlOGENOLOGY
(P < 0.05) PRL concentrations compared with controls (98.2 and 45.7 + 25.9 ngfml, respectively) and this pattern persisted throughout in samples obtained during the remaining 5 h. Thus, treatment with a single injection containing 5 mg of oPRL elevated serum PRL during a 6-h period immediately following the treatment. Because an oPRL by day of sampling interaction was not detected (P > 0.20) in samples obtained on alternate days during the treatment period, serum PRL response to exogenous oPRL was examined across the 28-d treatment period. Serum PRL in these pretreatment, prefeeding samples was similar (P > 0.10) in oPRL-treated (55.7 + 5.3 ng/ml) and control (48.3 + 5.3 ng/ml) ewe lambs. Therefore, thesedata imply that exogenous oPRc was apparently cleared rapidly from the circulatory system of the ewe, because administering 5 mg oPRL as a single injection on alternate days did not sustain serum PRL levels for the 48-h period between treatments. In contrast, other studies (27, 28) showed that administering exogenous oGH in a similar fashion to that reported in our study sustained elevated serum growth hormone concentrations during treatment and for an extended period of time after treatments were terminated. Serum Growth Hormone and Insulin Because oPRL by sampling hour interactions were not observed for serum growth hormone (P > 0.20) or insulin (P > 0.05) in intensive samples collected on Days 0 and 28, serum growth hormone and insulin
Table 3.
Serum growth hormone (GH) and insulin (ng/ml) in ewe lambs before and for 6 hours after ovine prolactin (oPRL) administration on days 0 and 28 of the 28-day treatment period
Item
oPRL, mglalternate day Before treatment", After treatmentL -t 0 5 SEM" 0 5 SEMI
Day Oe GH Insulin
4.0 1.8
3.8 1.3
0.7 0.3
3.4 1.2
3.3 0.9
0.2 0.2
Day 28e GH Insulin
3.3 0.9
3.2 0.6
0.5 0.1
3.0 0.5
3.2 0.5
0.2 0.1
*Treatments were administered on alternate days for 28 d before bbreeding; five ewes (0 mg oPRL) and six ewes (5 mg oPRL). Values based on a single sample from each animal immediately before treatments were administered. 'Values represent pooled means from samples collected at 30-min intervals for 6 h after treatment on each day (oPRL by sampling time, dP > 0.20 for GH and P > 0.05 for insulin). Based on five ewe lambs/treatment. eRow values within day do not differ (P > 0.10).
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THERIOGENOLOGY responses to exogenous oPRL were examined across the 6-h sampling Serum growth hormone and insulin period on each day (Table 3). concentrations were similar (P > 0.10) between treatment groups immediately before oPRL was administered on both days. Furthermore, exogenous oPRL had no effect (P > 0.10) on serum growth hormone profiles for 6 h after treatment on Days 0 and 28. Likewise, serum insulin profiles were comparable (P > 0.10) between control and oPRL-treated ewe lambs on both Days 0 and 28. Thus, 5 mg of exogenous oPRL did not appear to affect secretion of other metabolic hormones since circulating levels of growth hormone and insulin were unaltered. Plaut et al. (29) reported similar findings in lactating dairy cows treated with 120 mg bovine PRLfday during two, 14-d treatment periods following parturition. Reproductive Performance Ovarian cycliclty was determined by quantifying serum progesterone in jugular samples obtained during treatment and breeding. An estrous cycle was judged to have occurred if serum samples collected on two consecutive sampling days contained more 1.0 ng/ml of than progesterone. No differences (P > 0.30) in the percentage of ewe lambs cycling during the 28-d treatment period were detected between groups (63.6 and 50.0% for control and oPRL-treated ewe lambs, respectively). Likewise, the percentages of ewe lambs cycling during the breeding season were similar (P > 0.30) between groups (100 and 91.7% for control and oPRL-treated ewe lambs, respectively). Subsequent lambing percentages were comparable (P > 0.30) between groups, with 36.4% of control and 25.0% of oPRL-treated ewes producing offspring. Additionally, all ewes lambing produced single offspring. Reproductive performance of ewe lambs is generally poor and ewe lambs display lower lambing rates than mature ewes (2, 30). Furthermore, Dyrmundsson (2) has suggested that these findings may result from the failure of ewe lambs to reach proper stages of physiological and endocrinological development necessary for normal sexual function during their first year. Results from the present study support these findings and suggest that administering 5 mg of oPRL on alternate days for 28 d before breeding does not enhance growth, stimulate early cyclicity or improve reproductive performance of virgin fine-wool ewe lambs.
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Davis, S. L., Hill, K. M., Ohlson, D. L. and Jacobs, J. A. Influence of chronic thyrotropin-releasing hormone injections on secretion of prolactin, thyrotropin and growth hormone and on growth rate in wether lambs. J. Anim. Sci. %:1244-1250 (1976).
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Bauman, D. E. and McCutcheon, S. N. The effects of growth hormone and prolactin on metabolism. In: L. P. Milligan, W. L. Grovum and A. Dobson (Eds). Controlof Digestion and Metabolism in Ruminants. Prentice-Hall Press, Englewood Cliffs, NJ, 1986, pp. 436-455.
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Forbes, J. M., Driver, P. M., Brown, W. B., Scanes, C. G and Hart, I. c. The effect of daylength on the growth of lambs. 2. Blood growth hormone, prolactin, insulin and concentrations of thyroxine, and the effect of feeding. Anim. Prod. 29:43-51 (1979).
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Eisemann, J. H., Bauman, D. E., Hogue, D. E. and Travis, H. F. Evaluation of a role for prolactin in growth and the photoperiodinduced growth response in sheep. J. Anim. Sci. -59:86-94 (1984).
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Peel, C. J., Bauman, D. E., Gorewit, R. C. and Sniffen, C. J. Effect of exogenous growth hormone on lactational performance in high yielding dairy cows. J. Nutr. -111:1662-1671 (1981).
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Hallford, D. M., Hudgens, R. E., Morrical, D. G., Schoenemann, H. Kiesling. Ii.E. and Smith, G. S. Influence of short-term conM suiption of sewage solids on productivity of fall lambing ewes and performance of their offspring. J. Anim. Sci. -54:922-932 (1982).
Influence of suckling status 18. Hoefler, W. C. and Hallford, D. M. and type of birth on serum hormone profiles and return to estrus Theriogenology early-postpartum spring-lambing ewes. in -27:887-895 (1987). 19.
Sanson, D. W. and Hallford, D. M. Growth response, carcass characteristics and serum glucose in lambs fed tolazamide. Rep. Int. -29:461-471 (1984).
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Swanson, L. V., Hafs, H. D. and Morrow, H. D. Ovarian characteristics and serum LH, prolactin, progesterone and glucocorticoids from first estrus to breeding size in Holstein heifers. J. Anim. Sci. -34:284-293 (1972).
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Holcombe, D. W., Hallford, D. M. and Hoefler, W. C. Reproductive and lactational responses and serum growth hormone and insulin in fine-wool ewes treated with ovine growth hormone. Anim. Prod. 46:195-202 (1988). -
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SAS Institute Inc.
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ABSTRACT Twenty-four 6-mo-old ewe lambs received one of two ovine prolactin (oPRL) treatments 28 d before fall breeding. Beginning on the first day of treatment (Day 0), 12 lambs received a subcutaneous injection (12 ml) of a carrier vehicle (0 mg oPRL) on alternate days for 28 d while 12 lambs received injections containing 5 mg oPRL. On Days 0 and 28, jugular blood was collected from six lambs in each group before treatment and at 30-min intervals for 6 h thereafter. Neither feed intake, efficiency of gain nor animal weights differed (P > 0.20) between groups. One hour after treatment on Day 0, ewe lambs receiving 5 mg oPRL had greater (P < 0.10) serum PRL levels than did controls (121.9 and 61.5 + 24.7 ng/ml, respectively). Differences in serum PRL persisted throughout remaining sampling intervals on both Days 0 and 28. Serum samples obtained on alternate days during the 28-d treatment period revealed no differences (P > 0.20) in PRL concentrations between control (48.3 + 5.3 ng/ml) and oPRL-treated (55.7 + 5.3 ng/ml) ewes. Neither serum-insulin nor growth hormone respondyd (P > 0.05) to exogenous oPRL on either Day 0 or 28. No difference (P > 0.30) in percentage of ewe lambs cycling during treatment or breeding was detected between groups. Subsequent lambing percentages were similar (P > 0.30), with 36.4% of control and 25.0% of oPRL-treated ewes producing offspring. Administering 5 mg oPRL on alternate days for 28 d before breeding did not enhance growth and(or) reproductive performance in virgin ewe lambs. Key words:
sheep, prolactin, reproduction, growth hormone, insulin
Acknowledgments: Appreciation is expressed to D. Holcombe, W. Hoefler, R. Oyler and R. Reynolds. We also thank those who supplied assay materials: National Hormone and Pituitary Program (Univ. of Maryland School of Medicine), Dr. A. F. Parlow (Pituitary Hormones and Antisera Center, Harbor/UCLA Lilly Laboratories Research Medical Center, Torrance, CA), (Indianapolis, IN; oInsulin, lot 615-1112B-108-I) and Dr. G. D. Niswender (Department of Physiology and Biophysics, Colorado State yniversity, Fort Collins, CO; progesterone-11 BSA antiserum 1337). Journal Article 1425 of the New Mexico Agricultural Experiment Station. Reprint requests and correspondence to D. M. Hallford.
JULY 1989 VOL. 32 NO. 1
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THERIOGENOLOGY INTRODUCTION Improving reproductive efficiency is a major goal of sheep producers. Although not commonly practiced, breeding ewe lambs is one method that may increase lifetime productivity and longevity of ewes (1, 2). However, reproductive performance in ewe Iambs is generally poor compared with mature ewes because of failure to attain puberty (2), shorter duration of estrus (3), lower lambing rates (4) and higher incidences of early embryonic mortality (5) and general reproductive failures (6). Body size and age appear to be critical factors influencing the onset of puberty in ewe lambs (2, 7). Lambs growing at faster rates generally exhibit earlier estrus and are more likely to conceive at younger ages (4, 8). Methods of accelerating growth rates that do not limit reproduction might, therefore, benefit overall reproductive performance of the ewe lamb. Recent evidence indicates that prolactin (PRL) may be involved in the regulation of overall body growth and nutrient partitioning (9). Research suggests PRL is an anabolic hormone in ruminants (10 - 12) and numerous treatments which increase growth rates also elevate serum PRL (13). Additionally, interactions between PRL and growth have been suggested by studies involving photoperiodic changes. Beneficial gains associated with various photoperiod regimens have been reported in rams (11, 14) and wethers (15) in the presence of elevated PRL concentrations. Thus, distinct roles for PRL in growth have yet to be defined in domestic animals. This study examined effects of exogenous ovine PRL on growth responses, endocrine profiles and reproductive performance of fine-wool ewe lambs.
MATERIALS AND METHODS Thirty-three days before the fall breeding (5 d before initiating treatments), 24 fine-wool (Debouillet x Rambouillet) ewe lambs (6 mo old) were randomly allotted to one of two treatments (12 lambs/group). Lambs in each group were placed in one of four 3.1 x 9.1 m pens (eight pens total, three lambs/pen) to allow determination of feed intake and efficiency of weight gain during a 28-d study. Throughout the study, lambs were allowed ad libitum access to a pelleted alfalfa diet, water, salt, mineral and shade. Average diet composition (dry matter basis) was 93.0% dry matter, 15.6% crude protein, 9.1% ash, 2.3% ether extract, 1.2% calcium and 0.3% phosphorus. Weights were obtained before treatment and at 10-d intervals thereafter, at the end of breeding and near the time of lambing. Beginning on Day 0 (first day of treatment), ewe lambs received subcutaneous injections containing either 0 or 5 mg oPRL (NIDDK-oPRL-17) on alternate days for 28 d. Ovine PRL was solubilized in 0.9% saline and adjusted to pH 9.6 by addition of 0.025 M NaHC03 and 0.025 M Na2C0 (16), such that the final volume (12 ml) contained either 0 or 5 mg OPaL. Jugular blood samples were collected by venipuncture from all ewes on alternate days (beginning on Day 0) throughout treatment and every third day (beginning on Day 29) during breeding to determine the onset of estrual activity. On Days 0 and 28 (after a 12-h fast and another
46
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THERIOGENOLOGY feeding for 1 h), jugular blood samples were collected from six ewes in each treatment group before treatment and at 30-min intervals for 6 h thereafter to examine acute and chronic effects of oPRL on serum hormone profiles. Blood was allowed to clot at room temperature for 45 min, after which serum was separated by centrifugation (2,300 x g, 4"C, 15 min) and stored (-ZO'C) until serum hormones could be quantified by radioimmunoassay. Serum progesterone (17), growth hormone (18) and insulin (19) were determined by previously validated procedures. The PRL assay utilized rabbit anti-oPRL (NIDDK-anti-oPRL-1), the specificity of which has been previously reported (Technical Report 109, Pituitary Hormones and Antisera Center, Harbor UCLA Medical Center, Torrance, CA). Radioiodination of highly purified oPRL (NIDDK-oPRL-I-2) was accomplished by the chloramine odium ?I!? metabisulfite method described by Niswender et al. (20) except I was used as the tracer. Standards were prepared by making appropriate dilutions of oPRL (NIDDK-oPRL-17) in 0.01 M phosphate buffered saline containing 1% bovine serum albumin (PBS + 1% BSA, pH 7.0). Anti-rabbit gamma globulin was raised in sheep (second antibody) using procedures described by Swanson et al. (21) and was suspended at a working dilution (1:lO) in 0.01 M PBS containing 0.05 M ethylenediaminetetraacetic acid (PBS-EDTA, pH 7.0). The assay was accomplished by pipetting standard or serum (usually 0.025 to 0.04 ml) fnto 12 x 75-m disposable plastic culture tubes and then normalizing all tubes to 0.5 ml with PBS + 1% BSA. Rabbit anti-oPRL (0.2 ml of a l:lOO,OOO dilution in PBS-EDTA + 1:400 normal153bbit serum, pH 7.0) was added to each tube followed by addition of I-oPRL (0.1 ml PBS + 1% BSA containing approximately 25,000 cpm). Tubes were vortexed briefly and incubated overnight at 4'C, followed by addition of 0.2 ml of second antibody and a second overnight incubation (4°C). Following final incubation (third day), 1.0 ml cold (4°C) 0.01 M PBS was added and tubes were centrifuged (2,300 x g, 4°C) for 15 min. After decanting the supernate, radioactivity in the bound fraction was determined and hormone concentrations were calculated. Under these assay condfs3ons, a l:lOO,OOO dilution of anti-oPRL routinely bound 51% of added I-oPRL, of which 3.8% was nonspecific binding. Addition of 1.0 ng oPRL to the resulted in approximately a 56% reduction in the amount of BfSay I-oPRL bound to the primary antibody. When 25 ng oPRL were added to ovine serum containing 7.5 nglml, 33.7 + 0.5 ng/ml (mean c SEM) were recovered (104%). Within- (n = 12) and between- (n = 3) assay coefficients of variation were 9.7 and 10.8X, respectively. Following the 28-d treatment period, ewe lambs were placed in a single pen (4.3 x 15.2 m) and joined with fertile Debouillet rams for 34 d. One control ewe became ill during breeding and her data were subsequently eliminated, with the exception of feed intake and efficiency of weight gain, which were determined on a pen basis. Feed intake and efficiency of weight gain were evaluated by analysis of variance for completely random designs (22). Animal weights and serum hormone profiles were subjected to split-plot analysis of variance (23) with effects of oPRL treatment included in the main plot and tested using animal within treatment as the error term. Categorical data (cyclicity and reproductive performance) were
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THERIOGENOLOGY analyzed using Chi-square procedures (24). Analyses of variance and categorical analyses were computed using the General Linear Models and Catmod Procedures, respectively, of the Statistical Analysis System (25).
RESULTS AND DISCUSSION Feed Intake and Ewe Weights Total feed intake (pen basis) during the 28-d treatment period was similar (P > 0.20) between groups, with ewe lambs receiving 5 mg oPRL consuming (mean + SEM) 161.9 + 8.3 kg compared with 153.3 + 8.3 kg for control animals, Likewise, ewe weights were comparable-(P > 0.10) throughout the 28 d of treatment with oPRL-treated ewes weighing 47.9 + 1.1 kg on the final day of treatment (Day 28) compared with 47.5 + 1.i kg for the controls (Table 1). Furthermore, when total feed intak; and total weight gain (pen basis) were examined in terms of efficiency of gain, no differences were detected (P > 0.10) between groups (10.4 and 9.4 + 0.4 kg feed/kg gain for ewe lambs receiving 0 and 5 mg oPRL, respectively). Thus, administering 5 mg oPRL to ewe lambs on alternate days for 28 d did not influence intake, gain or efficiency of gain. Likewise, Eisemann et al. (15) reported that daily administration of exogenous oPRL for 9 wk did not enhance total gain or feed intake in wether lambs maintained in 8 h light:16 h dark compared with controls kept under similar conditions. However, active immunization against PRL (26) or inhibition of PRL secretion with 2-Br-a-ergocryptine (15) in rams and wethers resulted in decreased body weight gains and feed intake. Ewe weights were also obtained at regular intervals following the 28-d treatment period. Weights were similar (P > 0.10) between control and oPRL-treated ewe lambs at the end of breeding (50.1 and 51.8 + 1.2 kg, respectively) as well as near the time of parturition (55.3 and 57.6 2 1.2 kg, respectively). Table I.
Body weights (kg) of ewe lambs Eeceiving ovine prolactin (oPRL) for 28 d before breeding
Item No. of animals
oPRL, mglalternate d 0 5 11
12
41.7 43.5 45.7 47.5 44.6
42.0 43.2 46.0 47.9 44.8
SEMC
Time after initiating treatment (days) 0 9 19 28 Overall meanb
1.0 1.1 1.1 1.1 1.0
*Split-plot analysis of variance revealed no oPRL by day interaction b(P > 0.50); therefore overall means were tested. Row values do not differ (P > 0.10). 'Based on 11 ewe lambs/treatment.
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THERIOGENOLOGY Serum Prolactin In jugular samples obtained immediately before treatment and at 30-min intervals for 6 h after treatment on Days 0 and 28, split-plot analyses of variance revealed oPRL by sampling time interactions (P < 0.10); therefore, effects of exogenous oPRL were examined within sampling hour on each respective day (Table 2). Also, because PRL values varied widely, ranks rather than actual hormone values were analyzed on both days. Serum PRL did not differ (P > 0.10) before and 30 min after treatment on either Day 0 or 28 in control and oPRL-treated ewe lambs. However, by 1 h after treatment on Day 0, ewe lambs receiving 5 mg oPRL had greater (P < 0.10) serum PRL values than did controls (121.9 and 61.5 + 24.7 ngfml, respectively). Throughout the remaining sampling intervals, oPRL-treated ewe lambs generally maintained two- to three-fold higher PRL concentrations than those observed for controls. Similar responses were detected on Day 28. Within 1 h after treatment, oPRL-treated ewe lambs had higher
Table 2.
Serum prolactin (ngfml) in ewe lambs before and for six hours after ovine prolactin (oPRL) administration on days 0 and 28 during a 28-day treatment perioda
Time (hours)
0
5
,oPRL, mgialternate day k Day 28 SEM' 0 5
SEM'
Before treatment 0
90.7h
94.gh
31.1
39.zh
34.Zh
10.6
After treatment 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
87.7h 61.5d 63.5d 56.1d 70.4h 45.1f 61.3h 49.4d 47*4f 58.0f 50.Zf 46.7d
131.0h 121.ge 118.1e 100.6e 115.6h 84.4g 97.gh 86.5e 109.11 151.48 156.2' 125.ge
23.0 24.7 19.8 17.4 23.4 12.4 15.1 13.3 26.4 39.9 39.5 31.3
65.gh 45.7f 62.5d 41.gf 55.1f 31.Zf 38.Zf 24.1f 52.gf 39.6f 28.Zf 37.2f
115.gh 98.Zg 93.ze 103.48 128.3' 106.3' l08.4g 71.6g 116.4g 104.5g 94.3g 105.58
39.4 25.9 11.9 17.8 29.9 17.7 12.5 6.5 22.2 9.6 12.2 13.4
"Five ewes (0 mg oPRL) and six ewes (5 mg oPRL); sampled at 30-min intervals for 6 h after treatment. Treatments were administered b(~.~.) on alternate days for 28 d before breeding. Ovine prolactin by sampling hour interaction was detected (P < 0.10); therefore, treatment effects were examined within hour. Because PRL values varied widely, ranks were analyzed within =time of sampling. Analyses were conducted within sampling day. d ,Based on 5 ewe lambs/treatment. ' Row values within day differ (P < 0.10). f'gRow values within day differ (P < 0.05) hRow values within day do not differ (P ; 0.10).
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THERlOGENOLOGY
(P < 0.05) PRL concentrations compared with controls (98.2 and 45.7 + 25.9 ngfml, respectively) and this pattern persisted throughout in samples obtained during the remaining 5 h. Thus, treatment with a single injection containing 5 mg of oPRL elevated serum PRL during a 6-h period immediately following the treatment. Because an oPRL by day of sampling interaction was not detected (P > 0.20) in samples obtained on alternate days during the treatment period, serum PRL response to exogenous oPRL was examined across the 28-d treatment period. Serum PRL in these pretreatment, prefeeding samples was similar (P > 0.10) in oPRL-treated (55.7 + 5.3 ng/ml) and control (48.3 + 5.3 ng/ml) ewe lambs. Therefore, thesedata imply that exogenous oPRc was apparently cleared rapidly from the circulatory system of the ewe, because administering 5 mg oPRL as a single injection on alternate days did not sustain serum PRL levels for the 48-h period between treatments. In contrast, other studies (27, 28) showed that administering exogenous oGH in a similar fashion to that reported in our study sustained elevated serum growth hormone concentrations during treatment and for an extended period of time after treatments were terminated. Serum Growth Hormone and Insulin Because oPRL by sampling hour interactions were not observed for serum growth hormone (P > 0.20) or insulin (P > 0.05) in intensive samples collected on Days 0 and 28, serum growth hormone and insulin
Table 3.
Serum growth hormone (GH) and insulin (ng/ml) in ewe lambs before and for 6 hours after ovine prolactin (oPRL) administration on days 0 and 28 of the 28-day treatment period
Item
oPRL, mglalternate day Before treatment", After treatmentL -t 0 5 SEM" 0 5 SEMI
Day Oe GH Insulin
4.0 1.8
3.8 1.3
0.7 0.3
3.4 1.2
3.3 0.9
0.2 0.2
Day 28e GH Insulin
3.3 0.9
3.2 0.6
0.5 0.1
3.0 0.5
3.2 0.5
0.2 0.1
*Treatments were administered on alternate days for 28 d before bbreeding; five ewes (0 mg oPRL) and six ewes (5 mg oPRL). Values based on a single sample from each animal immediately before treatments were administered. 'Values represent pooled means from samples collected at 30-min intervals for 6 h after treatment on each day (oPRL by sampling time, dP > 0.20 for GH and P > 0.05 for insulin). Based on five ewe lambs/treatment. eRow values within day do not differ (P > 0.10).
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THERIOGENOLOGY responses to exogenous oPRL were examined across the 6-h sampling Serum growth hormone and insulin period on each day (Table 3). concentrations were similar (P > 0.10) between treatment groups immediately before oPRL was administered on both days. Furthermore, exogenous oPRL had no effect (P > 0.10) on serum growth hormone profiles for 6 h after treatment on Days 0 and 28. Likewise, serum insulin profiles were comparable (P > 0.10) between control and oPRL-treated ewe lambs on both Days 0 and 28. Thus, 5 mg of exogenous oPRL did not appear to affect secretion of other metabolic hormones since circulating levels of growth hormone and insulin were unaltered. Plaut et al. (29) reported similar findings in lactating dairy cows treated with 120 mg bovine PRLfday during two, 14-d treatment periods following parturition. Reproductive Performance Ovarian cycliclty was determined by quantifying serum progesterone in jugular samples obtained during treatment and breeding. An estrous cycle was judged to have occurred if serum samples collected on two consecutive sampling days contained more 1.0 ng/ml of than progesterone. No differences (P > 0.30) in the percentage of ewe lambs cycling during the 28-d treatment period were detected between groups (63.6 and 50.0% for control and oPRL-treated ewe lambs, respectively). Likewise, the percentages of ewe lambs cycling during the breeding season were similar (P > 0.30) between groups (100 and 91.7% for control and oPRL-treated ewe lambs, respectively). Subsequent lambing percentages were comparable (P > 0.30) between groups, with 36.4% of control and 25.0% of oPRL-treated ewes producing offspring. Additionally, all ewes lambing produced single offspring. Reproductive performance of ewe lambs is generally poor and ewe lambs display lower lambing rates than mature ewes (2, 30). Furthermore, Dyrmundsson (2) has suggested that these findings may result from the failure of ewe lambs to reach proper stages of physiological and endocrinological development necessary for normal sexual function during their first year. Results from the present study support these findings and suggest that administering 5 mg of oPRL on alternate days for 28 d before breeding does not enhance growth, stimulate early cyclicity or improve reproductive performance of virgin fine-wool ewe lambs.
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SAS Institute Inc.
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