Recombinant bST Research Articles

Production of Bst polymerase for diagnosis of different infections using loop-mediated isothermal amplification

Introduction. The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) possesses 5'-3' DNA polymerase activity, 5'-3' displacement activity and high processivity. These properties make it possible to use Bst DNA polymerase in loop-mediated isothermal amplification (LAMP), which provides highly specific amplification of the target sequence and is used for rapid detection of agents causing human infectious diseases.
 The purpose of the study was to produce a recombinant Bst polymerase enzyme in the bacterial expression system and to assess its properties for LAMP-based diagnostics of infectious diseases.
 Materials and methods. Expression constructs carrying the Bst polymerase gene were obtained using genetic engineering techniques. Different Escherichia coli strains were used for protein expression. Metal-chelate and gel filtration chromatography techniques were used for protein purification. Catalytic characteristics of the enzyme were assessed in loop-mediated isothermal amplification reactions using AmpliSens SARS-CoV-2-IT, AmpliSens IAV-IT and AmpliSens IBV-IT diagnostic systems designed for high-quality detection of SARS-CoV-2, influenza A virus (IAV) and influenza B virus (IBV) RNA, respectively.
 Results. The offered protocol for production, extraction and purification of recombinant Bst polymerase makes it possible to produce the enzyme in the bacterial expression system using E. coli cells in a soluble form and reaching the yield up to 20% of the total cell mass. In LAMP reactions, the obtained enzyme demonstrates activity comparable with that of the commercial enzyme Bst 2.0 (NEB).
 Conclusion. Considering the fast purification and production of the enzyme, the obtained recombinant Bst polymerase can be used in LAMP-based diagnostic kits.

Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.

Tracing (r)bST in cattle: Liquid-based options for extraction and separation

ABSTRACTGrowth hormone (GH) or somatotropin (ST) is a species-specific polypeptide hormone produced in the pituitary gland of vertebrates. When administered exogenously to dairy cattle, it has galactopoietic effects and is capable of increasing the milk yield. The beginning of the commercial production of recombinant variants of bovine somatotropin (rbST), by the end of the 20th century, had a major economic impact in the dairy industry. Recombinant bST enabled large-scale applications in farms, enhancing significantly milk production. While it is banned in the European Union (EU), several countries permit the trade and use of recombinant somatotropins in animal husbandry. Also, rbST-free dairy products can be frequently found in the market of those countries, even though these labels are not actually verified in a laboratory. In this context, effective analytical methods are needed for residue control to avoid an illegal use of rbST but also to prevent fraudulent labeling in some cases. The present review includes studies published in the last 5 years (from 2012 to 2017) to monitor rbST in bovine animals, using liquid-based applications. It is then intended to serve as a practical guide to help those laboratories interested in developing analytical methods to detect rbST use and abuse.

Effects of feeding rumen-degradable valine on milk production in late-lactating dairy cows

The study objective was to determine if feeding the rumen-degradable AA Val can increase milk production comparable to recombinant bovine somatotropin (bST). Eight multiparous late-lactating (255±26.4d in milk) Holstein dairy cows were blocked by milk yield (34.1±8.25kg/d) and randomly assigned to 1 of 4 treatments in a replicated 4×4 Latin square design with 21-d periods (7d for dietary adaptation and 14d for data collection). Treatments were control (CON), a single injection of recombinant bST (rbST), and Val fed at 40 (V40) and 80g/d (V80). Cows were fed a total mixed ration with a distillers dried grains carrier at 113.4g/d containing none or added AA. Dry matter intake (21.3, 22.0, 22.8, and 21.5kg/d for CON, rbST, V40, and V80, respectively) was similar among treatments, except cows receiving V40 had greater dry matter intake than cows receiving V80. Milk yield (22.0, 26.1, 25.2, and 24.9kg/d), 3.5% fat-corrected milk (22.1, 25.4, 24.4, and 24.3kg/d), and energy-corrected milk (22.7, 26.1, 25.1, and 24.9kg/d) were increased at similar amounts for cows receiving rbST, V40, and V80 compared with CON cows. Milk fat percentages (3.51, 3.36, 3.32, and 3.38%) were greatest for CON cows compared with cows receiving V40, whereas cows receiving other treatments were intermediate and similar. Milk protein percentages (3.20, 3.12, 3.15, and 3.13%) were greater for CON cows compared with cows receiving rbST and V40, whereas cows receiving V80 were intermediate and similar. Ruminal isobutyrate (1.19, 1.24, 1.44, and 1.74 mol/100 mol) concentrations were increased for cows receiving V40 and V80 compared with CON and rbST cows, with cows receiving V80 having greater concentrations than cows receiving V40. Plasma growth hormone concentrations (1.78, 1.99, 1.55, and 1.45 ng/mL) were greater for cows receiving rbST compared with cows receiving V40 and V80, whereas CON cows were intermediate and similar. Plasma insulin-like growth factor-1 concentrations (60.4, 106.1, 65.9, and 58.3 ng/mL) were greater for cows receiving rbST compared with cows receiving other treatments. This study suggests that feeding rumen degradable Val can increase milk yield comparable to recombinant bST.

PCR-based Gene Synthesis, Cloning, Expression, Purification and Characterization of Bst DNA Polymerase in E. coli Cells

Gene synthesis is a technique for modifying genes for studying gene function, structure and expression. There are several PCR based strategies and non-PCR based strategies for gene synthesis. Here we perform the 2 step gene synthesis combining assembly PCR and sequential overlap extension PCR strategies to synthesize the full length of Bst DNA polymerase (2,648bp) and express the protein in E. coli cells. The full length Bst DNA polymerase was divided into 5 short DNA Fragments and the oligonucloetides were designed for the entire sequence each with 40-45 mers. In step 1, the oligonuleotides of each fragment were assembled and then the gene fragment was amplified separately through assembly PCR method. In step 2, sequentially the two adjacent fragments were assembled through overlap extension PCR at a time until the full length gene completely synthesized and finally cloned into pCR®2.1-TOPO vector. Then the full length Bst DNA pol gene was subcloned into pET28a(+) expression vector and finally expressed in BL21(DE3) E. coli cells. The purified protein was identified by MALDITOF analysis. The polymerization activity of the recombinant Bst DNA polymerase was compared with commercial enzyme. The 98kDA recombinant Bst DNA polymerase succeed in amplification of 100bp DNA fragment via helicase dependent amplification (HDA).

Serum hormone profiles, pregnancy rates, and offspring performance of Rambouillet ewes treated with recombinant bovine somatotropin before breeding1,2

An experiment was conducted to examine effects of bovine ST (bST) on serum hormone concentrations, pregnancy rates, and offspring performance. Before initiation of a fall breeding period, 75 Rambouillet ewes (68.8 ± 1.5 kg) received an intravaginal insert containing 0.3 g of progesterone (P4) to synchronize onset of estrus. After 12 d, inserts were removed (d 0), and ewes (stratified by BW and age) received either 0 (control, n = 37) or 250 (n = 38) mg of recombinant bST (Posilac, Monsanto, St. Louis, MO, subcutaneously). Ewes were joined with fertile rams 24 h after insert removal. Blood samples were collected from 12 ewes in each treatment group daily from d 0 to 20 after insert removal. Serum IGF-I concentrations were 315 and 437 (± 58) ng/mL in control and bST-treated ewes 2 d after receiving bST (P = 0.02) and remained increased (P < 0.03) in bST-treated ewes throughout the 13-d period (P < 0.05). Serum prolactin (P > 0.10) and estradiol (P = 0.65) were similar between treatments. Serum triiodothyronine (T3) and thyroxine (T4) concentrations were similar (P > 0.20) between treatments from d 0 through 8. Controls had greater (P < 0.04) serum T3 and T4 concentrations than treated ewes did until d 18. Serum P4 was similar (P > 0.10) in control and bST-treated ewes from d 0 through 3 but was increased (P < 0.05) from d 4 to 8 in control ewes. Serum P4 was again similar (P > 0.10) between treatments from d 9 to 20. Serum insulin concentrations were 0.44 and 1.74 (± 0.19) ng/mL in control and bST-treated ewes, respectively, 1 d after receiving bST (P < 0.001) and remained increased (P < 0.03) in bST-treated ewes through d 9 (P < 0.03). Serum glucose was increased (P = 0.003) from d 0 to 10 in bST-treated ewes compared with controls. Thirty-three of 37 (89%) control ewes were pregnant, whereas 27 of 38 (71%) bST-treated ewes were pregnant (P = 0.05). As a percentage of ewes lambing, 61% and 39% of control ewes produced single and twin lambs, respectively, compared with 41% and 59% of bST-treated ewes (P = 0.12). Lamb 60-d adjusted weaning weights were 23.0 and 21.2 (± 0.65) kg for offspring produced by control and bST-treated dams, respectively (P = 0.04). In conclusion, serum IGF-I, insulin, and glucose were greater whereas serum T3, T4, and P4 were less in bST-treated ewes than in controls. Pregnancy rates and offspring adjusted weaning weights were decreased by bST treatment immediately before breeding.

Survey of the fatty acid composition of retail milk differing in label claims based on production management practices

Consumers are becoming increasingly health conscious, and food product choices have expanded. Choices in the dairy case include fluid milk labeled according to production management practices. Such labeling practices may be misunderstood and perceived by consumers to reflect differences in the quality or nutritional content of milk. Our objective was to investigate nutritional differences in specialty labeled milk, specifically to compare the fatty acid (FA) composition of conventional milk with milk labeled as recombinant bST (rbST)-free or organic. The retail milk samples (n=292) obtained from the 48 contiguous states of the United States represented the consumer supply of pasteurized, homogenized milk of 3 milk types: conventionally produced milk with no specialty labeling, milk labeled rbST-free, and milk labeled organic. We found no statistical differences in the FA composition of conventional and rbST-free milk; however, these 2 groups were statistically different from organic milk for several FA. When measuring FA as a percentage of total FA, organic milk was higher in saturated FA (65.9 vs. 62.8%) and lower in monounsaturated FA (26.8 vs. 29.7%) and polyunsaturated FA (4.3 vs. 4.8%) compared with the average of conventional and rbST-free retail milk samples. Likewise, among bioactive FA compared as a percentage of total FA, organic milk was slightly lower in trans 18:1 FA (2.8 vs. 3.1%) and higher in n-3 FA (0.82 vs. 0.50%) and conjugated linoleic acid (0.70 vs. 0.57%). From a public health perspective, the direction for some of these differences would be considered desirable and for others would be considered undesirable; however, without exception, the magnitudes of the differences in milk FA composition among milk label types were minor and of no physiological importance when considering public health or dietary recommendations. Overall, when data from our analysis of FA composition of conventional milk and milk labeled rbST-free or organic were combined with previous analytical comparisons of the quality and composition of these retail milk samples, results established that there were no meaningful differences that would affect public health and that all milks were similar in nutritional quality and wholesomeness.

On the alterations in serum concentration of somatotropin and insuline-like growth factor 1 in lactating cows after the treatment with a little studied recombinant bovine somatotropin

A study was performed to delineate bST and IGF-1 variation, over a whole lactation, in cows treated with a nowadays widely commercialised but little studied sustained release formulation of recombinant bST. Total bST levels were found to be exceptionally high in the first days after administration, but decreased rapidly in the second week after injection. The increase in the IGF-1 serum concentration was significant for almost the entire biweekly cycle. Based on this study, the peaks of ST (often above 100 ng/ml) are considered particularly unlikely to be found in non-treated bovines, even under pathological conditions, especially when detected in a number of animals within a herd. Notwithstanding the great heterogeneity of results on this topic, these data suggest that tests against fraud involving the use of rbST in dairy products may be regarded as a feasible possibility.

Effect of Increased Milking Frequency in Early Lactation With or Without Recombinant Bovine Somatotropin

Multiparous Holstein cows (n=300) were assigned to 1 of 2 milking frequency treatments at parturition. Cows were either milked 6 times (6×) or 3 times (3×) daily to determine effects on early lactation milk yields and subsequent lactation persistency with or without use of recombinant bST (rbST). Treatments included a control group milked 3× and 3 groups milked 6× for either the first 7, 14, or 21 days in milk (DIM). Those 4 groups of cows all received rbST starting at 63 DIM. The fifth treatment group was also milked 6× for the first 21 DIM but those cows received no rbST during the entire lactation. All cows returned to 3× milking after their respective treatment periods ended. Cows milked 3× tended to produce more milk (43.2 vs. 41.5 and 41.0±1.1kg/d) during the first 9 wk of lactation compared with cows milked 6× for 7 or 21 DIM, respectively. Group milk yields after wk 9 averaged 38.3±0.7kg/d and did not differ among various groups assigned to an increased milking frequency in early lactation. Percentages of milk fat (3.8±0.12%) and protein (2.9±0.06%) did not differ among treatments during the first 9 wk after calving. Early lactation milk yield (41.9±1.2kg/d) did not differ between the 2 groups of cows milked 6× for 21 DIM. However, cows subsequently administered rbST (at 63 DIM) produced more milk (38.8 vs. 34.2±0.9kg/d) from wk 10 to 44. The number of cows sent to the hospital during the 305-d trial for mastitis (97), digestive disorders (14), respiratory issues (9), lameness (22), or retained placenta (16), were not affected by treatments (χ2=0.49). Under the conditions of this commercial dairy herd in Arizona, increasing milking frequency to 6 times daily for 7 to 21 d at the start of lactation conditions did not increase milk yield nor improve lactation persistency.

Effects of Dexamethasone and Growth Hormone Treatment on Hepatic Gluconeogenic Enzymes in Calves

The hypothesis was tested that dexamethasone (DX) and bovine somatotropin (bST) alter expression or activity of gluconeogenic enzymes in neonatal calves. Holstein dairy calves (n=24) were randomly divided in 4 groups and were treated with saline (control group), with DX at 30μg/kg body weight per d (CDX), with 500mg of sustained-release recombinant bST every 14 d (CbST), and with the combination of DX and bST from d 3 through 42 of life (CbSTDX). Plasma glucose and insulin concentrations were elevated throughout the study in CbSTDX, and insulin concentrations were elevated in CDX from d 7 to 28. Treatment with DX and the combination of DX and bST increased plasma glucagon concentrations from d 14 to 42, but decreased plasma cortisol concentrations on d 7 and 14 when compared with control calves. In liver, phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels were reduced in CDX and CbSTDX when compared with control calves or CbST. The activity of PEPCK on d 14 was higher in CbSTDX compared with control calves. Pyruvate carboxylase mRNA levels were decreased on d 7 in CDX and CbSTDX. Pyruvate carboxylase activities on d 14 and 28 were lower in CDX and CbSTDX than in control calves or CbST. These data indicate an age-dependent response to DX for blood metabolites, expression and activities of hepatic PEPCK and pyruvate carboxylase, and for effects of bST, suggesting that glucocorticoid status is important.

Development and Validation of a Short-Term, Serum-Free Culture System for Bovine Granulosa Cells: Evaluation of the Effects of Somatotropin and Growth Hormone-Releasing Factor on Estradiol Production

The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5mm in diameter), medium (6 to 14mm), or the largest (6 to 19mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5α-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol>progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P<0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells from all follicle classifications was increased by FSH, but FSH only enhanced estradiol production by granulosa cells from the largest estrogen-active follicles on d 2 to 5. Recombinant bST blocked the FSH-induced increase in estradiol by granulosa cells from the largest estrogen-active follicles on d 2 to 5, whereas rbGRF had no effect on steroid production. Based on these results, we concluded that short-term, serum-free culture of bovine granulosal cells obtained from first-wave follicles at an abattoir could be used to reflect reliably the original in vivo estradiol-producing capacity of granulosal cells, and that neither rbST nor rbGRF enhance basal or FSH-induced estradiol production by bovine granulosa cells from first-wave follicles.

Effect of recombinant bovine somatotropin on milk production and composition of cows with Streptococcus uberis mastitis.

The protective effect of bovine somatotropin (bST) during experimental Streptococcus uberis mastitis in cows was studied. The left quarters of 10 cows were infected with 500 cfu of S. uberis O140J. Five cows were subcutaneously treated with 500 mg of recombinant bST 7 d before and after infection, and 5 control cows received the excipient. In the treated cows, total milk production significantly increased after the first and second bST treatments. After infection, milk production decreased 24 and 40% in the infected quarters, 6 and 14% in the uninfected quarters, and 15 and 28% overall for treated and control cows, respectively. In the bST group, milk production was completely restored after 3 wk, but, in the control group, total production and the production of the infected quarters remained lower than preinfection production. The increase in somatic cell count occurred earlier and more rapidly in the control group, and the return to normal values was also more rapid in these cows. The amount of bacteria in milk was higher in the control cows. Changes in milk composition, such as lactose, protein, fat, Na+, K+, and Cl-, were significantly more pronounced in the control cows. Also, clinical symptoms were more prominent in the control cows. Somatotropin protected the mammary gland from excessive production losses and compositional changes during a subsequent episode of experimentally induced Streptococcus uberis mastitis and significantly improved the normalization of production and composition, which indicates a beneficial effect on the restoration of the integrity of the blood-milk barrier.

Effect of bovine somatotropin on neutrophil functions and clinical symptoms during Streptococcus uberis mastitis.

The effect of recombinant bovine somatotropin (bST) on the chemiluminescence, diapedesis, and expression of adhesion receptors (CD11a, CD11b, CD18) of isolated polymorphonuclear leukocytes was studied. The plasma concentrations of insulin-like growth factor-I (IGF-I), bST, cortisol, and alpha-lactalbumin were also monitored. In addition, general and local clinical symptoms and the differentiation of circulating leukocytes were also studied during experimentally induced Streptococcus uberis mastitis in cows. Ten cows were infected with 500 cfu of S. uberis O140J in both left quarters. Five cows were subcutaneously treated with 500 mg of recombinant bST 7 d before and after infection, and 5 control cows received the excipient. General (fever, tachycardia, inappetance, and depression) and local symptoms (swelling, pain, firmness, and flecks in milk) were more acute, severe, and longer-lasting in control cows. Treatment with bST had no effect on chemiluminescence and diapedesis of circulating polymorphonuclear leukocytes and no effect on the expression of adhesion receptors. Recombinant bST induced significantly higher IGF-I and bST concentrations in plasma. The leukopenia observed after infection was less pronounced in the bST-treated cows, and the number of circulating band neutrophils and metamyelocytes was significantly lower in the treated group. The concentration of cortisol did not differ between both groups, but the blood concentration of alpha-lactalbumin significantly increased in both groups from 6 d after infection. These results showed that treatment with recombinant bST improves animal welfare by protecting the cows from severe local and general clinical symptoms during subsequent S. uberis mastitis, but that it has no effect on chemiluminescence, diapedesis, and the expression of adhesion receptors of circulating polymorphonuclear leukocytes.

Primary structure, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus.

The complete primary structure of inorganic pyrophosphatase [EC 3.6. 1.1] from Bacillus stearothermophilus (ATCC 12016) was determined at the amino acid level by automated Edman degradation. The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18,796. The amino acid sequence of the enzyme is almost identical to that of thermophilic bacterium PS-3. Based on the determined primary structure, a PCR-amplified semi-synthetic gene was constructed and expressed in Escherichia coli JM109. The recombinant Bst. PPase showed the same characteristics and activity as the authentic enzyme, and exhibits higher thermostability than the E. coli enzyme. Furthermore, we prepared tyrosine-substituted variants by site-directed mutagenesis to elucidate the role of two highly conserved tyrosines (Y46 and Y130). As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected. However, the wild-type and two variants exhibited different thermostability behaviors in the presence or absence of Mg2+. Therefore, these tyrosines may contribute to the structural integrity of the active site of the enzyme.

Biology of somatotropin in growth and lactation of domestic animals.

Impressive progress has been made during the past 15 years in our understanding of the biology of somatotropin (ST) in domestic animals. In part, this progress was sparked by advances in biotechnology that made feasible the production of large quantities of recombinant bovine ST (bST) and porcine ST (pST). The availability of recombinant bST and pST resulted in an exponential increase in investigations that explored their role in growth and lactation biology, as well as evaluated their potential for commercial use. Collectively, these studies established that administration of bST to lactating dairy cows increased milk yield, and treatment of growing pigs with pST markedly stimulated muscle growth and reduced fat deposition. In addition to these "efficacy" studies, a substantial number of investigations examined the mechanisms by which ST affects lactation and growth of domestic animals. This review summarizes the diverse physiological effects ST has on growth and lactation and discusses the underlying mechanisms that mediate these effects in domestic animals.

Agricultural Biotechnology and the Environment: Science, Policy, and Social Issues By S. Krimsky and R. P. Wrubel (review)

Agricultural Biotechnology and the Environment: Science, Policy, and Social Issues. By S. KRiMSKYand R. P. Wrubel. Urbana: Univ. of Illinois Press, 1996. Pp. 294. 47.50; Paper 18.95 The fact that controversies over the environmental implications of agricultural biotechnology have been so prominent seems somewhat ironic. As Krimsky and Wrubel stress in Chapters 1 and 11 ?? Agricultural Biotechnology and the Environment, agriculture is a fairly minor sector ofcommercial biotechnology, accounting for only 17.7 percent of private investment in the early 1990s, or less than a quarter of the amount invested in diagnostics and therapeutics. In addition to agriculture being small potatoes in the world ofbiotechnology, agricultural biotechnology involves the utilization of "natural" processes, which should allow these technologies to be more environmentally benign than the chemical inputs they are presumably replacing. As if to anticipate these objections, Krimsky and Wrubel note on the first page of the book, " [I]nevitably, the questionwill be, Whatwas all the fuss aboutbiotechnology?" Krimsky and Wrubel's Agricultural Biotechnology and the Environment does an admirable job at explaining "the fuss." Their book is the most comprehensive social analysis to date of the reasons for and complexities of the environmental impacts and implications ofagricultural biotechnologies. The book has three major components . The first is introductory material (Introduction and Chapter 1), which provides background on theory and empirical research on agricultural research and technological change. The heart of the book consists of nine case-study chapters on particular biotechnology products. The selection of case studies is comprehensive and appropriate, including: biotechnology products of historical significance (i.e., die "ice-minus" Pseudomonas that largely defined the early politics of regulation of GEOs) ; all of the major agricultural biotechnology products now in the pipeline that are likely to have significant environmental implications or impacts (especially bST, herbicide-tolerant crop varieties, Bt-engineered crops) ; transgenic plant products and "identity-preserved" crops (e.g., Flavr Savr tomato); biotechnologies with future promise but uncertainty of future success (e.g., transgenic nitrogen -fixing crops and bacteria); and combinations of the preceding categories (e.g., transgenic livestock for "transgenic pharming" vs. food production). The concluding material (Chapter 11 and Conclusion) provides an overview of the issues and evidence on agricultural biotechnology and the environment. Each of the case-study chapters contains a terse but useful history of the development of the technology and of the debate and evidence regarding environmental and other impacts. The core scientific literatures on each technology are identified relatively well, though the coverage of the critical or dissident literature is generally better than that of the conventional or promotional literatures. For better or worse, "environment" is employed in a broad way, with human health implications figuring very prominently in many of the chapters. The chapters are also excellent at teasing out potentially important indirect environmental effects of particular biotechnology products, as well as the more obvious direct ones. A good example concerns recombinant bST. While promoters of the technology claim that bST will have environmental benefits (because it will reduce the cow population and involve less land under cultivation and less manure) , Krimsky and Wrubel demonstrate that it could have the opposite effect. Because bST is a capital-intensive technology that is most suitable for larger farms, the remaining cows will be concentrated on larger (often "factory") dairy farms, where manure disposal and pollution can be more 310 Book Reviews serious problems. And the cropland "released" from feeding dairy cows will notjust lie fallow. It will likely be shifted from legumes and grasses to more environmentally destructive monocultural row crops. The essence of Krimsky and Wrubel's argument is that while biotechnology need not have negative environmental impacts, several of these technologies are very likely to do so. In the authors' view, while biotechnology has some potential to substitute for chemicals, the overall thrust of the first generation of agricultural biotechnologies is that they are being developed to be compatible with intensive, chemically based agriculture. Also, most of these biotechnologies were developed through a "technology push" process; rather than being "demand-pull" technologies that respond to a social need, agricultural biotechnologies tend to be solutions in search of problems. The textbook example of this is said to be...

Effect of Recombinant Bovine Somatotropin and Calcium Salts of Long-Chain Fatty Acids on Milk from Italian Buffalo

Fifty-one lactating Italian river buffalo were used in an 84-d study to evaluate the effects of recombinant bovine somatotropin (bST) and Ca salts of long-chain fatty acids on productive performance. Treatments were 1) control diet, 2) the control diet plus 0.3 kg/d of added Ca salts of long-chain fatty acids, 3) the control diet plus 320mg of recombinant bST injected every 21 d for four cycles, and 4) the control diet plus 0.3 kg/d of added Ca salts of long-chain fatty acids and 320mg of recombinant bST administered as previously described. Administration of bST and Ca salts of long-chain fatty acids increased milk production. Milk fat percentage was not affected by treatments. The percentage of short-chain fatty acids in milk fat was reduced by the addition of Ca salts. Medium-chain, long-chain, and unsaturated fatty acids in milk fat were increased by bST treatment. Milk protein percentage was decreased by the addition of Ca salts of long-chain fatty acids. Milk casein content, as a percentage of total protein or as a percentage of true protein, was unaffected by bST. Body condition score was lowered by bST administration, but the addition of Ca salts of long-chain fatty acids reduced body condition loss in buffalo that were treated with somatotropin.

Effects on Nutrient and Hormonal Profile of Long-Term Infusions of Glucose or Insulin Plus Glucose in Cows Treated with Recombinant Bovine Somatotropin Before Peak Milk Yield

Ten Holstein cows were treated with 30.9mg·d−1 of recombinant bST from 15 to 41 d of lactation. The Latin square design included three infusion periods of 6 d each with 3 d of rest between infusion periods. Infusions were physiological saline, glucose (50g·h−1), and insulin plus glucose (12.5IU·h−1 + 50g·h−1). Blood was collected continuously during the last 24h of each infusion period. Statistical analyses of data for energy balance, milk yield, and DMI were performed on the last 3 d of each infusion period. Production data before and after infusions (i.e., no recombinant bST) estimated that recombinant bST increased milk yield of cows infused with glucose and saline by 3.1 and 3.6kg·d−1, respectively. Net energy intake was not affected by infusion, but glucose infusion resulted in higher BW loss than did saline infusion (2.33 vs.0.08kg·d−1, respectively), and insulin plus glucose infusion resulted in BW gain (0.65kg·d−1). Milk yield was 39.9, 39.6, and 37.6kg·d−1 for cows infused with saline, glucose, and insulin plus glucose, respectively. The insulin plus glucose infusion increased milk protein 11 and 14% compared with response to saline and glucose infusions, respectively; no change occurred in the proportion of casein and whey proteins. Serum bST was increased 109% with exogenous recombinant bST. Serum IGF-I was lower for cows infused with glucose than for those infused with saline (21.03 vs. 27.44ng·ml−1) and increased to 46.55ng·ml−1 for cows infused with insulin plus glucose. Serum concentrations of insulin and glucose were 13.7 and 56.7, 18.5 and 61.9, and 30.5μIU·ml−1 and 39.4mg·dl−1 for cows infused with saline, glucose, and insulin plus glucose, respectively.The results of this study suggest that low concentrations of plasma insulin in early lactation may limit the IGF-I response to recombinant bST (uncoupling). Despite higher IGF-I, milk yield was lower, probably as a result of low blood glucose. These results suggest that, in early lactation, insulin is still anabolic because the BW gain of cows increased. However, milk yield was still higher than that for cows in late lactation with similar insulin concentrations.

Effect of Slow-Release Somatotropin on the Pattern of Milk Yield Between and Within Injection Intervals

Thirty-six Alpine goats (10 primiparous and 26 multiparous) were used to study the milk yield response induced by recombinant bST administered in a sustained-delivery vehicle. Eighteen goats were injected with recombinant bST at 4-wk intervals from about 7 wk postpartum for 12 wk. Patterns of milk yield were highly affected by injection of recombinant bST. Milk yield of treated goats increased from day of injection, peaked between d 3 to 5 after treatment, and then decreased progressively. This pattern of response was well fitted by a curve containing two exponential terms: Yi = A × [exp (–k1di – exp (–k2di)], where Yi = daily relative response of treated goats to recombinant bST, computed as a deviation of least squares means of milk yield at day i (i = 0, …, 26) of the treatment interval from least squares means of milk yield at d 27 of the treatment interval; A = constant; k1 and k2 = response rate parameters; and di = day from treatment (i = 0, …, 27). Administration of recombinant bST also improved persistency of lactation, and the overall milk yield response to exogenous hormone was 300 g/d (13.9%) higher than the yield of untreated goats. This response was due to both the short-term response immediately after injection (53%) and the medium-term effect on lactation persistency.

Legislative and Legal Restrictions on Labeling Information Regarding the Use of Recombinant Bovine Somatotropin

With the issuance of the “Interim Guidance on the Voluntary Labeling of Milk and Milk Products from Cows That Have Not Been Treated with Recombinant Bovine Somatotropin” by the FDA in February 1994, the Monsanto Company, Inc. (St.Louis, MO) commenced the commercial sale of Posilac®. Because of farmer and consumer concerns, marketing organizations, state administrative agencies, and state legislatures responded with various voluntary and mandatory regulations and rules for labeling milk and milk products with information regarding the use of recombinant bST. A regulatory labeling framework that varies from state to state has caused problems for some marketing organizations. Individuals and organizations may now turn to the judicial arena as an avenue to challenge unfavorable developments.