Stig Purup

High body weight gain and reduced bovine mammary growth: physiological basis and implications for milk yield potential

Available evidence concerning the relationship between growth rate, mammary growth and milk yield in heifers leads to these conclusions: 1) Increased growth rate due to high feeding level before puberty onset can lead to reduced pubertal mammary growth and reduced milk yield potential. 2) Increased growth rate due to high feeding level after puberty and during pregnancy have no effect on mammary growth and milk yield. 3) Higher body weight gain due to higher genetic potential for growth is positively related to milk yield. The negative effect of high feeding level before puberty occurs in all breeds, but the level of feeding causing reduced yield varies. Variation in responses between experiments suggests that feeding regimes that support high growth rates without negative effect on yield can be developed. A breakthrough most likely will originate from increased knowledge of the physiological relationship between nutrition and mammary development. Our investigations suggest that blood growth hormone (GH) is important for mammary development, and that the negative effect of high feeding level on mammary development may be due to reduced blood GH. GH, however, does not bind to mammary tissue. Experiments with exogenous GH suggest that GH acts on mammary tissue via IGF-I, but IGF-I is increased by high feeding level - not decreased as GH. This paradoxical relationship cannot be explained by changes in circulating IGF binding proteins. However, the sensitivity of mammary tissue to IGF-I is reduced by high feeding level, probably due to the action of locally produced binding proteins and/or growth factors.

Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro.

Abstract Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.

Familial Hypercholesterolemia and Atherosclerosis in Cloned Minipigs Created by DNA Transposition of a Human PCSK9 Gain-of-Function Mutant

A transgenic pig model of familial hypercholesterolemia can be used for translational atherosclerosis research. A Model of We hope to inherit our parents’ good features, like blue eyes or musical talent, but not their high cholesterol. Familial hypercholesterolemia, which is passed down in families, results in high levels of “bad” cholesterol [low-density lipoprotein (LDL)] and early onset of cardiovascular disease. To further translational research in this area, Al-Mashhadi and coauthors created a large-animal model of this genetic disease, showing that these pigs develop hypercholesterolemia and atherosclerosis much like people do. The D374Y gain-of-function mutation in the PCSK9 gene (which is conserved between pig and human) causes a severe form of hypercholesterolemia and, ultimately, atherosclerosis. Al-Mashhadi and colleagues engineered transposon-based vectors to express D374Y-PCSK9. After confirming function in human liver cancer cells, the authors cloned minipigs that expressed the mutant gene. On a low-fat diet, these pigs had higher total and LDL cholesterol than their wild-type counterparts. Breeding the male transgenic pigs with wild-type sows produced offspring that also had higher plasma LDL levels compared with normal, healthy pigs. A high-fat, high-cholesterol diet induced severe hypercholesterolemia in these animals as well as accelerated development of atherosclerosis that has human-like lesions. Other large-animal models only develop hypercholesterolemia when placed on the right diet, and small-animal models cannot recapitulate human-like pathology. The PCSK9 transgenic pigs created by Al-Mashhadi et al. develop hypercholesterolemia even on low-fat diets, and thus reflect the inherited human disease. This large-animal model will be important for better understanding the pathogenesis of familial hypercholesterolemia and for testing new therapeutics and imaging modalities before moving into human trials. Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5 nM TSA for 22-24 h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15 h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.

Effects of Clover-Grass Silages and Concentrate Supplementation on the Content of Phytoestrogens in Dairy Cow Milk

A 2 x 2 factorial continuous experiment was conducted with 28 Norwegian Red dairy cows in early lactation to compare milk content of phytoestrogens when feeding ad libitum white clover (WCS) or red clover (RCS) grass silages prepared from the second and third cut without and with 10 kg/d supplementation of a standard concentrate. The cows were offered either RCS or WCS for 88 d (period 1) and thereafter a mixed red clover-white clover-grass silage for 48 d (period 2). Total dry matter intake and milk yield were not affected by forage type but increased with concentrate supplementation. Intake of isoflavones was several times greater in RCS than in WCS, whereas intake of lignans was greater in WCS. Concentrate supplementation reduced the intake of most phytoestrogens. Compared with WCS, RCS diets yielded milk with a greater content of flavonoids, whereas milk from WCS diets had greater contents of the mammalian lignans enterodiol and enterolactone. The content of the isoflavan equol was particularly high in RCS diets. There was no apparent carryover effect of clover type on milk phytoestrogen content because there was no difference in content between the silage treatments 3 wk after the cows were transferred to the same silage diet (period 2). Concentrate supplementation reduced the milk contents of the flavonoids equol, biochanin A, and daidzein and increased the content of mammalian lignans. The effects of silage type and concentrate supplementation on milk contents of the individual phytoestrogens were related to the intake of the compound or its precursor, except for the effect of concentrate on mammalian lignans, for which the intake of the known precursors was also reduced. Overall, this study shows that feeding cows with silage containing red clover increases the milk content of flavonoids at both low and high concentrate supplementation levels, and decreases the content of nonflavonoids such as mammalian lignans, when compared with silage containing white clover. The increased content of phytoestrogens in milk may be important when the health benefits of milk are studied.

Differential effects of falcarinol and related aliphatic C17-polyacetylenes on intestinal cell proliferation.

Quantitative major polyacetylenes of carrots (falcarinol and falcarindiol) and American ginseng roots (falcarinol and panaxydol) were isolated and tested in human intestinal epithelial cells of normal (FHs 74 Int.) and cancer (Caco-2) origin. A hormesis effect was seen for all isolated polyacetylenes when added to Caco-2 cells in concentrations ranging from 1 ng/mL to 20 μg/mL. The relative inhibitory potency was falcarinol > panaxydol > falcarindiol. No hormesis effect was observed when adding the polyacetylenes to FHs 74 Int. cells. Instead, an inhibitory growth response was observed above 1 μg/mL. The relative inhibitory potency was panaxydol > falcarinol > falcarindiol. Maximal inhibition at 20 μg/mL corresponded to approximately 95% and 80% inhibition of cell proliferation in normal and cancer cells, respectively. Combinations of falcarinol and falcarindiol added to normal and cancer cells showed a synergistic response for the inhibition of cell growth. Furthermore, the oxidized form of falcarinol, falcarinon, showed a significantly less growth inhibitory effect in intestinal cells of both normal and cancer origin; hence, a hydroxyl group at C-3 may be important for activity of falcarinol-type polyacetylenes. Extracts of carrots, containing different amounts of falcarinol, falcarindiol, and falcarindiol 3-acetate had significant inhibitory effects on both normal and cancer cell proliferation. In cancer cells, the extract containing the highest concentration of falcarinol tended to have the highest growth inhibitory effect, in accordance with a higher potency of falcarinol than falcarindiol. The present study demonstrates that aliphatic C17-polyacetylenes are potential anticancer principles of carrots and related vegetables and that synergistic interaction between bioactive polyacetylenes may be important for their bioactivity.

Local IGF-I Axis in Peripubertal Ruminant Mammary Development

The regulation of mammary growth and development in heifers is accomplished by complexinteractions of hormones, growth factors, and extracellular matrix molecules. Many of thesegrowth stimulators are believed to be locally produced in the mammary gland and to beaffected by developmental and nutritional status. Although estrogen and growth hormone areconsidered critical to pubertal mammogenesis, results summarized in this review suggest thatIGF-I6 and IGF binding proteins are especially important locally-produced growth regulatorsin peripubertal ruminants. This assertion is supported by studies of ovariectomized heifers, inwhich increased stromal IGFBP-3 and reduced IGF-I correspond with a failure of udderdevelopment. Similarly, reduced mammary development with overfeeding coincides withreduced mitogenic activity of mammary tissue extracts and altered concentrations of IGF-Iand IGFBPs. In vitro studies convincingly demonstrate that much of the mitogenic activity ofmammary extracts or serum can be attributed to IGF-I and that alterations in IGFBP-3 modulateits effectiveness. Thus by analogy to second messenger mechanisms of action for proteinhormones, local mammary-derived growth factors likely explain many of the effects attributedto the classic mammogenic hormones.

Biological activity of bovine milk on proliferation of human intestinal cells.

To evaluate the bioactivity of bovine milk from different stages of lactation on human intestinal tissue, a human fetal small intestinal cell line was used as a model system. Milk samples representing six stages of lactation: days 1, 2-3, 6-7 and weeks 12 and 24 after parturition, 1 week before drying off, and milk-like secretion from two stages of the dry period: 7 weeks and 3-4 weeks before expected calving, were collected from 64 Holstein Friesian cows. The whey fraction of the milk or milk-like secretion was added to the culture medium in concentrations ranging from 0.078% to 10%. The growth-promoting activity of whey was measured by determining the incorporation of [3H]thymidine into DNA for the last 24 h of the culture period. Whey fractions from all six stages of lactation stimulated growth of intestinal cells. The growth-promoting activity of colostrum or milk significantly decreased within the first week after calving. The growth-promoting activity in mature milk increased gradually during lactation to reach a level significantly higher than that obtained with colostrum. The growth-promoting activity of whey from milk-like secretion collected after drying off was lower than that of colostrum. Whey from different stages of lactation contained significantly different concentrations of TGF-beta1 (0.5-27 ng/ml) and TGF-beta2 (12-1219 ng/ml). However, neither the differences in TGF-beta1 and TGF-beta2, nor the differences in IGF-I and IGF-binding proteins could fully explain the differences in growth-promoting activity of colostrums or milk from different stages of lactation, suggesting that other factors were also involved. The present study showed that bovine milk contained a number of biologically active components that affected growth and development of human intestinal tissue. The results showed that the growth-promoting activity of colostrum and milk was dependent on the stage of lactation in accordance with previous results obtained with mammary epithelial cells. The changes in growth-promoting activity with stage of lactation were probably related to changes in concentrations of several growth factors.

Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum.

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

Growth hormone and mammary development

Classic studies in rodents conducted in the 1950s showed that growth hormone (GH) is essential for mammary development both in the pubertal phase and during pregnancy. Since then, a considerable number of experiments have been carried out in ruminants to investigate the role of GH for regulation of normal mammary development and to examine the possibility of enhancing mammary growth by administration of GH. The available evidence demonstrates that GH treatment stimulates mammary growth before puberty, but the data do not convincingly support the idea that the effect is translated into increased milk yield. GH treatment during late pregnancy seems to stimulate both mammary growth and milk yield during lactation. The limited data concerning the effect of GH on mammary growth during lactation indicate that mammary growth is unaffected by GH treatment in early lactation, whereas GH seems to increase the amount of mammary parenchyma in mid-lactation. The mechanism of action of GH remains a puzzle, but the effect of exogenous GH most likely involves insulin-like growth factor-I (IGF-I). Full understanding of the role of endogenous GH for regulation of normal mammary development requires more knowledge about the interaction between GH and IGF-I and the interplay between the GH-IGF-I axis and locally produced factors, including receptors, binding proteins, and growth factors.