Growth performance, endocrine, and metabolite responses of finishing hogs to porcine prolactin.

Prolactin, a member of the somatotropin-prolactin-placental lactogen gene family, increases feed intake and rate of weight gain in several species. To determine whether prolactin affects growth performance and carcass composition in swine, recombinant porcine prolactin (rpPRL) was administered to finishing hogs. Doses of 0, 2, 4, 8, and 16 mg of rpPRL/d and 4 mg of recombinant porcine somatotropin (rpST)/d were administered to groups of seven barrows and seven gilts initially weighing 75.0 +/- .2 kg for a 28-d period. Recombinant pPRL did not alter feed intake or growth rate or affect carcass composition. In addition, most growth-related blood variables did not change, although plasma IGF-I was increased in the 8 and 16 mg of rpPRL treatment groups. At slaughter, mammary development was apparent in rpPRL-treated gilts and was characterized by distended alveolar and ductal lumina and presence of secretory material. In rPST-treated hogs, feed intake was decreased 28% (P < .01), gain/feed was increased more in barrows than in gilts (59 vs 39%, treatment x sex interaction, P = .035), and growth rate was increased 22%, but in barrows only (treatment x sex interaction P = .005). Compared with those in control hogs, circulating concentrations of IGF-I, insulin, and glucose were 175, 311, and 22% higher, respectively, and of blood urea nitrogen were 62% lower in rpST-treated hogs (P < .05). These results suggest that rpPRL, at the doses administered, does not increase feed intake in finishing hogs in contrast to rats and other species.

The effect of recombinant bovine placental lactogen on induced lactation in dairy heifers.

The primary objective of this study was to determine whether bovine placental lactogen stimulated additional mammary growth as assessed by milk yield from a lactation induced by steroids. Pubertal, nonpregnant Holstein heifers (n = 23) were given daily subcutaneous injections of estradiol-17 beta (0.05 mg/kg) and progesterone (0.25 mg/kg) for 7 d to initiate mammary growth. Prolactin secretion was suppressed in all heifers via bromocriptine, which was administered until d 15. Heifers were treated with either placental lactogen (40 mg/d; n = 12) or water (control group; n = 11) for 18 d. Lactation was induced by daily injection of dexamethasone for 3 d and twice daily injections of recombinant bovine prolactin for 5 d starting on d 18. From 3 to 8 wk of lactation, milk yield of heifers treated with placental lactogen was numerically higher (22%) than the yield of control heifers, but the difference was not significant because of the high coefficient of variation. Daily injection of bovine somatotropin (d 57 to 66 of lactation) increased milk yield of both groups and stimulated a greater numerical increase in milk yield for heifers that were treated with placental lactogen. These results support the hypothesis that bovine placental lactogen is mammogenic and is one of the factors that regulates mammary growth during pregnancy.

Placental lactogen and somatotropin: hormone binding to the corpus luteum and effects on the growth and functions of the ovary in heifers.

The effects of recombinant bovine placental lactogen (rbPL) and recombinant bovine somatotropin (rbST) on development of ovarian follicles and CL were tested in heifers. Estrus (day = 0) was synchronized and heifers were treated (Days 0-21) with either saline (control; n = 7), rbST (25 mg/day; n = 6), or rbPL. (50 mg/day; n = 8). Blood was collected daily for analyses of progesterone, estradiol, ST, PL, and insulin-like growth factor (IGF)-I; ultrasound was performed daily for measurement of follicles and CL. PL in plasma (mean +/- SE; ng/ml) averaged 4.1 +/- 0.2 for rbPL-treated heifers, and ST in plasma (ng/ml) averaged 2.7 +/- 0.3 for rbST-treated heifers. IGF-I in plasma (ng/ml) was increased for rbST-treated (198 +/- 10; p < 0.001) and rbPL-treated (143 +/- 9; p < 0.06) heifers compared to controls (117 +/- 9). After Day 9 of the estrous cycle, heifers treated with rbPL had larger CL (p < 0.001) and more progesterone in plasma (p < 0.001) than controls, whereas rbST-treated heifers were intermediate for these measures. Largest follicles were decreased in size (mm) throughout the estrous cycle for rbPL-treated heifers (12.9 +/- 0.4) compared to controls (14.2 +/- 0.5; p < 0.06) or heifers given rbST (14.0 +/- 0.5; p < 0.11). After Day 17 (preovulatory period), concentrations of estradiol in serum (pg/ml) were decreased for rbST-treated (2.7 +/- 0.3; p < 0.01) and rbPL-treated (2.9 +/- 0.2; p < 0.02) heifers compared to controls (3.8 +/- 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of mammogenesis and lactogenesis by recombinant bovine placental lactogen in steroid-primed dairy heifers.

A model of induced lactation was modified to examine the effects of bovine prolactin (bPRL) and bovine placental lactogen (bPL) on mammary growth and differentiation. Thirty-two peripubertal, non-pregnant Holstein heifers were given daily s.c. injections of oestradiol (0.05 mg/kg) and progesterone (0.25 mg/kg) for 7 days to initiate mammary growth. Treatment with bromocriptine (40 mg/3 days) reduced serum PRL concentrations to approximately 25% of pretreatment levels, for the duration of the study. On the day following the last steroid injection, groups of eight heifers were given twice daily s.c. injections of either saline (negative control), recombinant bPRL (rbPRL; 80 mg/day) or recombinant bPL (rbPL; 80 and 160 mg/day) for 7 days. At the end of this period (day 15), growth and differentiation of the mammary glands were assessed. Treatment with rbPL increased total mammary DNA above control value by 50 and 60% for the 80 and 160 mg/day doses respectively. However, total DNA was not different for the control and rbPRL-treated groups. The blood serum concentration of alpha-lactalbumin was measured daily throughout the study and used as an index of mammary differentiation. Both rbPRL and rbPL stimulated mammary differentiation (i.e. induction of milk synthesis), although rbPRL appeared to be more potent than rbPL. These results indicate that rbPL is lactogenic in vivo and strongly suggest that bPL is a mammary mitogen.

Performance, clinical chemistry, and carcass responses of finishing lambs to recombinant bovine somatotropin and bovine placental lactogen.

Bovine placental lactogen (PL) is a partial somatotropin agonist in the cow and decreases urea nitrogen, indicating increased nitrogen retention. In the present study, the somatogenic effects of bovine PL (bPL; 4 and 8 mg/d) were compared with those of bovine somatotropin (bST; 4 and 8 mg/d) in finishing lambs. Measures of comparison included growth performance, carcass composition, and growth-related clinical chemistry traits. Although feed efficiency during the first 3 wk of treatment with bPL was improved by 14% (P < .05), feed efficiency for the full 6-wk treatment period did not differ from that of control lambs. Responsiveness to bPL may have been attenuated by high titer antibodies present after 2 wk of treatment. However, bPL also did not influence growth-related clinical chemistry traits during short-term (7 d) treatment, strongly suggesting that bPL was ineffective in finishing lambs at the doses tested. In contrast, bST improved 6-wk feed efficiency by an average of 17% (P < .05) and decreased feed intake by an average of 12% (P < .05). In addition, measures of carcass composition including longissimus muscle area, specific gravity of the rack, kidney and pelvic fat, and fat thickness demonstrated that bST, but not bPL, treatment decreased carcass fatness and increased carcass leanness. Treatment with bST, but not with bPL, affected IGF-I, insulin, glucose, and urea nitrogen in a dose-related manner. Thus, daily injections of bPL did not affect either performance or carcass quality, whereas performance and carcass responses of finishing lambs to bST were consistent with those reported by others.

Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.

Stimulation of milk yield and feed intake by bovine placental lactogen in the dairy cow.

A 6 x 6 Latin square design was used to test the effects of recombinant bovine placental lactogen on milk yield, milk composition, feed intake, and blood hormone and metabolite levels in nonpregnant lactating cows. The six treatments (5, 10, 20, and 40 mg/d of placental lactogen, water as negative control, and 20 mg/d of bST as positive control) were administered by subcutaneous injection twice daily for 9 d. Blood samples were taken during the last 5 d of the treatment period. The three highest doses of placental lactogen increased milk yield, and there was a linear dose effect, although placental lactogen was less potent than bST. Milk concentrations of lactose, protein, and fat were not altered by any of the treatments. Dry matter intake was increased by two of the doses of placental lactogen, but not by bST. Blood urea N concentration was decreased in a dose-dependent manner by placental lactogen and was also decreased by bST. Similarly, serum insulin-like growth factor-I was increased in a dose-dependent manner by placental lactogen and was also increased by bST. Plasma concentrations of NEFA and glucose were increased by bST, but placental lactogen had little or no effect on either of these parameters. Thus, placental lactogen appears to act, in part, as a weak somatotropin agonist; however, it also appears to have specific activities, e.g., stimulating feed intake.

Serum half-life and in-vivo actions of recombinant bovine placental lactogen in the dairy cow.

The clearance rate of recombinant bovine placental lactogen (rbPL) from the blood serum of four lactating dairy cows was measured using a specific radioimmunoassay. Two animals were non-pregnant, while the other two were at approximately 120 days of gestation. The rbPL was administered as an i.v. bolus injection (4 mg total) via an indwelling jugular catheter. Blood samples were taken periodically for 180 min and assayed for rbPL. Analysis of the clearance curves for the bolus injection suggested a single-compartment model and a serum half-life of 7.25 min. In a second experiment with the same animals, following cessation of lactation, rbPL or bovine GH (bGH) were administered by s.c. injection (50 mg/day) for 5 consecutive days. Blood samples were taken twice per day during the treatment period and a 3-day pretreatment period. Samples were analysed for glucose, blood urea nitrogen (BUN), non-esterified fatty acids (NEFA), creatinine, insulin, insulin-like growth factor-I (IGF-I) and IGF-II, tri-iodothyronine (T3), progesterone and IGF-binding protein-2 (IGFBP-2) to determine whether rbPL mediates similar metabolic effects to those of bGH. Administration of bGH stimulated an increase in NEFA, glucose, T3 and insulin, whereas none of these variables was affected by rbPL. The plasma concentrations of IGF-I and IGF-II were both increased by treatment with rbPL but, to a lesser extent than occurred with bGH. Interestingly, BUN and IGFBP-2 concentrations were reduced equally by bGH and rbPL. These results suggest that rbPL does not necessarily act as a GH agonist but, rather, may have distinct effects on intermediary metabolism that could be mediated through another specific receptor.

Stimulation of body weight gain of the mature female rat by bovine GH and bovine placental lactogen.

Mature female rats (200 g) were treated for 10 days with either recombinant bovine GH (bGH) or recombinant bovine placental lactogen (bPL) to compare the somatogenic responses elicited by these hormones. The treatments were administered by daily s.c. injection at four dose levels (0.19, 0.56, 1.67 and 5.0 mg/day). Both bGH and bPL stimulated significant increases in weight gain, but the slopes of the dose-response curves were different (P less than 0.05). Bovine PL was more potent than bGH (P less than 0.01) at the lowest dose, although there were no differences between treatment groups at the three higher doses. Feed consumption was stimulated more by bPL than bGH at all doses (P less than 0.001). The concentration of insulin-like growth factor-I (IGF-I) in blood plasma was increased by bGH in a dose-responsive manner and was higher than control at doses of 1.67 and 5 mg/day (P less than 0.05). Low doses of bPL stimulated increases in IGF-I similar to those with bGH. At the highest dose of bPL, however, there was no concomitant increase in plasma IGF-I. Nevertheless, the growth rate of the animals in this group matched that of the group given the highest dose of bGH. Receptor binding studies indicated that bPL bound to both GH and prolactin receptors. This is consistent with the growth data which suggests that bPL stimulated weight gain through a somatogenic mechanism as well as by another route, possibly mediated by lactogenic receptors.

A novel concatenated dimer of recombinant bovine somatotropin.

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized in Escherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.

Simplified method for enzymatic urine oxalate assay.

A procedure for the quantitative determination of oxalate in urine suitable for use in clinical laboratories is described. Oxalate is extracted from urine and subsequently assayed by measuring the amount of hydrogen peroxide produced in an oxidation reaction catalyzed by oxalate oxidase. The reproducibility of the method was assessed by within-run and day-to-day reproducibility studies and the accuracy of the method was assessed by determining the amount of oxalate recovered in spiked urine samples. The correlation obtained between the method described here and a reference method was 0.976.