Contrasting Size Distributions among the Wing Spots of a Pierid Butterfly Suggest Different Selective Histories.

Males of the small cabbage butterfly Pieris rapae crucivora have two dark or melanic spots in the central white area of each dorsal forewing, an anterior spot (aS) and a posterior spot (pS). We used characteristics of the size distributions of these spots to infer how selection has acted on them during their evolution. Our study reveals that the aS size distribution is normal while that of pS is very right-skewed. Moreover, aS size is larger and less variable than pS size. These results suggest that the aS has been under stabilizing selection while the pS has not. The context in which this selection is acting is not yet clear, nor is it clear why pS persists as a wing marking.

Effect of ruminal administration of soy sauce oil on rumen fermentation, milk production and blood parameters in dairy cows.

OBJECTIVE: To evaluate soy sauce oil (a by-product of making whole soybean soy sauce) as a new dietary lipid source, a large amount of soy sauce oil was administered into the rumen of dairy cows. METHODS: Four Holstein dairy cows fitted with rumen cannulae were used in a 56-day experiment. Ruminal administration of soy sauce oil (1 kg/d) was carried out for 42 days from day 8 to day 49 to monitor nutritional, physiological and production responses. RESULTS: Dry matter intake and milk yield were not affected by soy sauce oil administration, whereas 4% fat-corrected milk yield and the percentage of milk fat decreased. Although ruminal concentration of total volatile fatty acids (VFA) and the proportion of individual VFA were partially affected by administration of soy sauce oil, values were within normal ranges, showing no apparent inhibition in rumen fermentation. Administration of soy sauce oil decreased the proportions of milk fatty acids with a carbon chain length of less than 18, and increased the proportions of stearic, oleic, vaccenic and conjugated linoleic acids. Conjugated linoleic acid content in milk became 5.9 to 8.8 times higher with soy sauce oil administration. Blood serum concentrations of non-esterified fatty acid, 3-hydroxybutyric acid, total cholesterol, free cholesterol, esterified cholesterol, triglyceride and phospholipid increased with administration of soy sauce oil, suggesting a higher energy status of the experimental cows. CONCLUSION: The results suggest that soy sauce oil could be a useful supplement to potentially improve milk functionality without adverse effects on ruminal fermentation and animal health. More detailed analysis is necessary to optimize the supplementation level of this new lipid source in feeding trials.

Effects of dietary vitamin K3 supplementation on vitamin K1 and K2 (menaquinone) dynamics in dairy cows.

The effect of dietary vitamin K3 (VK3) on ruminant animals is not fully investigated. The aim of this study was to examine the effects of dietary VK3 on lactation performance, rumen characteristics, and VK1 and menaquinone (MK, or VK2) dynamics in the rumen, plasma, and milk of dairy cows. Eight Holstein dairy cows in late lactation periods were used in two crossover trials including a control (nontreatment) and a 50 or 200 mg/day (d) VK3 supplementation group. After 14 days, plasma, ruminal fluid, and milk were sampled and their VK1 and MKs contents were measured using fluorescence-high-performance liquid chromatography (HPLC). Milk production was unchanged after feeding 50 mg/day VK3 but marginally decreased after feeding 200 mg/day VK3. The molar ratio of propionate in ruminal fluid was significantly increased on feeding 200 mg/day VK3. Additionally, MK-4 concentrations significantly increased in both plasma and milk after VK3 feeding (50 and 200 mg/day). In ruminal fluid, MK-4 concentrations increased after 200 mg/day VK3 feeding. These results suggest that VK3 may be a good source of MK-4, the biologically active form of VK, in Holstein dairy cows during their late lactation periods. This study provides a basis for understanding the physiological role of VK in dairy cows.

Effect of vitamin K3 supplementation on immunoglobulin G concentration in colostrum of periparturient Holstein dairy cows.

This study was to examine the effects of dietary vitamin K (VK) 3 supplementation on immune-related substances in milk, oxidative stress indices in plasma and VK1, and menaquinone 4 (MK-4) in plasma and milk in periparturient dairy cows. Forty healthy perinatal Holstein-Friesian dairy cows were used in this study. Twenty-one animals were randomly selected and categorized into the VK3 supplemented (50 mg/day/head as VK3) group; the remaining 19 were categorized into the control group. On day 3 after calving, blood and milk were sampled, and their chemical components were determined. The VK3 supplemented group had significantly higher menaquinone 4 levels in plasma and milk on day 3 postpartum than the control group. In addition, there was a significant increase in the immunoglobulin G (IgG) level in milk. VK3 may be absorbed from the gastrointestinal tract and converted to MK-4, the biologically active form of VK, in the mammary gland and other tissues. It was thought that the increase in MK-4 level in plasma and milk induced an increase in the concentration of IgG in milk. VK3 supplementation to periparturient dairy cows may contribute to the production of colostrum with high concentrations of IgG and MK-4.

Serotonin-induced mate rejection in the female cabbage butterfly, Pieris rapae crucivora.

Virgin female cabbage butterflies, Pieris rapae crucivora, accept and mate with courting males, whereas mated females reject them and assume the "mate refusal posture". This study tested whether the biogenic amines, serotonin (5HT), dopamine (DA), and octopamine (OA), were responsible for this change in behavior. The results showed that 2-3-day-old virgin females fed with 5HT rejected courting males significantly more frequently compared with controls fed on sucrose. In contrast, the proportions of courting males rejected by virgin females fed with either DA or OA did not differ from sucrose-fed controls. Oral application of each amine resulted in significantly increased levels of the amine applied (or its metabolite) in the brain. The results strongly suggest that 5HT or a 5HT metabolite may be responsible for the post-mating change in behavioral response of 2-3-day-old virgin females to courting males. Similar effects of 5HT treatment were observed in 6-8-day-old virgin females, but in this case the results were only marginally different from the controls, suggesting that the effect may decline with increasing female age.

Effects of ruminal administration of soy sauce oil on functional fatty acids in the rumen, blood and milk of dairy cows.

OBJECTIVE: Soy sauce oil, a byproduct of whole soybean processing by the soy sauce industry, was evaluated as a source of linoleic acid for dairy cows for the purpose of manipulating the composition of milk. METHODS: Eight dairy Holstein cows fitted with rumen cannulas were used for ruminal administration of soy sauce oil for a 28-day period using a 4×4 Latin square study design with 4 doses (0, 200, 400, and 600 g soy sauce oil/d). RESULTS: Although dry matter intake and milk yield were not affected by soy sauce oil administration, ruminal concentrations of total volatile fatty acids and acetate were decreased, specifically at 600 g/d administration. While milk fat percentage was decreased with administration of soy sauce oil, proportions of linoleic, vaccenic and conjugated linoleic acids in the rumen, blood and milk were increased with increasing soy sauce oil dose. CONCLUSION: These results suggest that soy sauce oil feeding could be useful for improving milk functionality without adverse effects on animal production performance when fed at less than 400 g/d.

Cluster analysis to evaluate disease risk in periparturient dairy cattle.

Predicting periparturient disease risk is of immense value to the dairy industry. Periparturient diseases are interrelated with each other; however, predicting the onset risk of these diseases has predominantly been based on a single blood parameter for a single disease. This study examined a new diagnostic method to predict the risk of periparturient diseases. We conducted cluster analysis of multiple blood constituents from 20 Holstein cattle at 1 week post-partum, and the cattle were divided into two groups, A or B. We then compared the periparturient and early-lactation blood constituents of these groups. Group B had significantly higher 3-hydroxybutyric acid concentrations and were suspected to have subclinical ketosis. Group B also had significantly lower calcium concentrations, with a tendency for subclinical hypocalcemia. We also performed discriminant analysis using blood parameters at 1 week post-partum, which grouped the population into the same two groups as the cluster analysis based on three variables: inorganic phosphorus, calcium, and either phospholipids or total cholesterol. We further showed that these discriminant functions could be used to predict the risk of periparturient disease even before parturition. Our results indicate that cluster analysis with multiple blood constituents is useful for predicting periparturient disease risks.

Saturated fatty acids stimulate and insulin suppresses CIDE-A expression in bovine mammary epithelial cells.

Cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDE-A) was first identified by its sequence homology with the N-terminal domain of DNA fragmentation factor (DFF). CIDE-A negatively regulates the activity of uncoupling protein 1 (UCP1) in brown adipose tissue. CIDE-A and UCP1 mRNA were detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and lactating bovine mammary glands. Physiological concentrations of saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of CIDE-A mRNA in bMEC. Treatment with insulin (5-10 ng/ml) induced down-regulation of CIDE-A and UCP1. The expression levels of CIDE-A and UCP1 mRNA in bovine mammary glands at various stages of the lactation cycle were determined by quantitative RT-PCR analysis. CIDE-A mRNA expression at peak lactation (2 months after parturition) was significantly higher than at dry off and non-pregnancy but not late lactation. These results suggest that CIDE-A and UCP1 are regulated by insulin and/or fatty acids in mammary epithelial cells and lactating mammary glands, and thereby play an important role in lipid and energy metabolism.

Saturated fatty acids stimulate and insulin suppresses BMCP1 expression in bovine mammary epithelial cells.

Brain-specific uncoupling proteins such as uncoupling protein 4 (UCP4) and brain mitochondrial carrier protein 1 (BMCP1; also known as UCP5) were identified by computational analysis for expressed sequence tag and hybridization screening. Both were detected at the mRNA level by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and lactating bovine mammary glands. Physiological concentrations of saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate), induced up-regulation of BMCP1 mRNA in bMEC. Treatment with insulin induced down-regulation of UCP4 and BMCP1. These results suggest that UCP4 and BMCP1 are regulated by insulin and/or fatty acids in mammary epithelial cells and lactating mammary glands, and thereby may play an important role in lipid and energy metabolism.

Short-chain fatty acid signaling pathways in bovine mammary epithelial cells.

GPR41 and 43 have recently been identified as G-protein-coupled cell-surface receptors for short-chain fatty acids (SCFAs). Bovine orthologs of GPR41 and 43 (bGPR41 and 43) mRNA were detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and various lactation stages of bovine mammary gland. Acetate and propionate caused an increase in intracellular Ca(2+) concentrations in these cells that was blocked by the treatment with pertussis toxin (PTX). SCFAs significantly reduced forskolin-induced cAMP concentrations in these cells. The phosphorylation of mitogen-activated protein kinase (MAPK) p38 was selectively increased by SCFAs. The downstream substrate heat shock protein 27 (HSP27) was also phosphorylated by SCFAs at Ser-78 and -82, but not -15. These results suggest that bGPR41 mainly, but not bGPR43, mediate SCFA signaling in mammary epithelial cells and thereby plays some important role in mammary gland.

Inaccurate mate recognition as a mating strategy of a 'pioneer male'.

Heterosubspecific mating experiments were carried out between two subspecies of cabbage butterflies, British Pieris rapae rapae and Japanese P. rapae crucivora, to examine how accurately males recognize the mates. The two subspecies are different in that the wings of female P. rapae rapae reflect little UV light, whereas those of female P. rapae crucivora are strongly UV-reflective. The wing colouration of P. rapae crucivora involving UV is believed to be critical in mate recognition. The results showed that males of both subspecies displayed mating behaviours, to and copulated with, females of both subspecies. Furthermore, P. rapae crucivora males exhibited mating behaviours and attempted to copulate with females of Pieris melete with low UV reflectance which are critically different from P. rapae crucivora females with high UV reflectance. Based on these findings, we propose the "pioneer male" hypothesis, which argues that such inaccurate mate recognition may sometimes be selectively beneficial for males and thus an adaptive mating strategy. The "pioneer male" was discussed in terms of its possible role in the evolution.

Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells.

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca(2+) concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.

Regulation of hormone-sensitive lipase expression by saturated fatty acids and hormones in bovine mammary epithelial cells.

Hormone-sensitive lipase was firstly identified as an epinephrine-induced lipase in adipocyte. HSL mRNA was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and bovine lactating mammary gland. Saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of HSL mRNA in a time- and concentration-dependent manner in bMEC. Treatment with insulin (5-10 ng/ml), dexamethasone (50-250 nM) or GH (50 ng/ml) induced down-regulation of HSL. These results suggest that HSL was regulated by fatty acids and some hormones in mammary epithelial cells and thereby play an important role of lipid and energy metabolism.

Regulation of uncoupling protein 2 expression by long-chain fatty acids and hormones in bovine mammary epithelial cells.

Although mammary epithelial cells are known to synthesize and accumulate triacylglycerol (TAG) in order to produce milk lipid in the cytosol, lipid and energy metabolism is still not fully understood. In this study, we assessed the effects of long-chain fatty acid (LCFA) on the accumulation of cytosolic TAG and uncoupling protein (UCP) 2 in cloned bovine mammary epithelial cells (bMEC). LCFAs significantly raised the expression of UCP2 mRNA and the accumulation of TAG. We observed the rapid elevation in UCP2 shown at 6h after LCFA treatment. Insulin (5-50 ng/ml) or dexamethasone (500 nM) significantly suppressed the expression of UCP2 mRNA. These results suggest that UCP2 play an important role of lipid and energy metabolism in mammary epithelial cells.

Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line.

The expression of GPR41 and 43, which have recently been identified as G-protein-coupled cell-surface receptors for short-chain fatty acids (SCFAs), was detected in a human breast cancer cell line (MCF-7) by RT-PCR. Acetate, propionate and butyrate induced an increase in intracellular Ca2+ in these cells that was not blocked by treatment with pertussis toxin (PTX). SCFAs significantly reduced forskolin-induced cAMP levels in these cells. The phosphorylation of mitogen-activated protein kinase (MAPK) p38 was selectively increased by SCFAs. The downstream substrate heat shock protein 27 (HSP27) was also phosphorylated by SCFAs at Ser-78 and-82, but not-15. Propionate induced elevations in intracellular Ca2+ and the phosphorylation of p38 were inhibited by the silencing of GPR43 using a specific siRNA. These results suggest that GPR41 and 43 mediate SCFA signaling in mammary epithelial cells and thereby play an important role in their stress management.

Geographic variation in ultraviolet reflectance of the wings of the female cabbage butterfly, Pieris rapae.

The British and Japanese subspecies of the cabbage butterfly, Pieris rapae , differ in terms of the UV reflectance of their wings ( Obara and Majerus, 2000 ). We studied the biogeographical distribution of the female cabbage butterfly having wings with UV reflectance around the Eurasian continent, and between Britain and Japan. For the study, we collected specimens from various locations. A gradient in the UV reflectance of the wings appears to exist along the west-east axis; reflectance was higher toward the east and reached a peak in butterflies in Japan. The UV-reflecting Japanese subspecies Pieris rapae crucivora was found exclusively along the east coast of the Eurasian continent. This suggests that the Japanese subspecies has evolved from a continental ancestor, with females having UV-absorbing wings. We discuss the results of our study with regard to the evolution and adaptive significance of UV coloration in the Japanese subspecies.

Mate preference in males of the cabbage butterfly, Pieris rapae crucivora, changes seasonally with the change in female UV color.

We initially investigated whether females of the cabbage butterfly, Pieris rapae crucivora, exhibit a seasonal change in ultraviolet wing color, which is a key stimulus for mate recognition by conspecific males, and whether and how a seasonal change affects the mating behavior of the males. We found that female UV wing color changes seasonally, the color being more pronounced in summer than in spring or autumn. We also demonstrated that male mate preference changes seasonally, concomitantly with the change in female UV color. Specifically, males appearing in summer exhibit a mating preference for summer-form females over spring- or autumn-form females, while those appearing in spring or autumn exhibit no seasonal preference, thereby facilitating more effective mate location. Our results suggest that this field of study will require more strictly controlled experimental investigation in which the seasonal change in UV color is considered when UV-influenced mating behaviors such as mate choice are investigated.

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells.

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.

Visfatin is present in bovine mammary epithelial cells, lactating mammary gland and milk, and its expression is regulated by cAMP pathway.

Visfatin was originally identified as a growth factor for immature B cells, and recently demonstrated to bind insulin receptor. Visfatin mRNA and protein were detected by RT-PCR and Western blot analysis in cloned bovine mammary epithelial cells, lactating bovine mammary gland and human breast cancer cell line, MCF-7. Immunocytochemical staining localized the visfatin protein in the cytosol and nucleus of both cells. Quantitative-RT-PCR analysis revealed that the expression of the visfatin mRNA was significantly elevated when treated with forskolin (500 microM), isopreterenol (1-10 microM) and dibutyric cyclic AMP (1 mM) for 24 h, and significantly reduced when treated with insulin (5-50 ng/ml) and dexsamethasone (0.5-250 nM) for 24 h. These results indicate that mammary epithelial cells express the visfatin protein and secrete them into the milk.

Oral administration of uridylic acid increases plasma leptin, but suppresses glucose and non-esterified fatty acid concentrations in rats.

Nucleic acids have been known to have biological effects on the digestive and immune systems, although less attention has been paid to the action on metabolism. In the present study, in order to investigate the effects of oral ingestion of uridylic acid (5'-uridine monophosphate, 5'-UMP) on hormonal and metabolic levels, we measured changes in the plasma concentrations of leptin, insulin, glucose, non-esterified fatty acids (NEFA), weights of the liver and abdominal fat and fat accumulation in the liver and M. gastrocnemius in male rats. Intragastric administration of 5'-UMP via a stomach tube at a dose of 44 mg/day for 7 days slightly (P=0.098) blunted the body weight gain without causing a significant change in food intake. The administration significantly reduced the plasma concentrations of glucose (P=0.004) and NEFA (P=0.004), whereas it significantly increased (P=0.03) plasma leptin concentration. The weights of perirenal (but not epididymal) fat (P=0.083) and the liver (P=0.061) were slightly increased. The triacylglyceride concentration in M. gastrocnemius was slightly increased (P=0.097), although the muscle weight was not significantly changed (P=0.197). In summary, acute oral administration of 5'-UMP was effective in the rat in reducing plasma concentrations of glucose and NEFA, an effect that was accompanied by an elevated plasma leptin concentration.