Cleaning procedures and cleanliness assessments of bucket milkers and suckling buckets on Japanese dairy farms.

Cleanliness of milking equipment is known to be important for the safety of dairy products and to prevent the spread of diseases among cows. We investigated the cleaning procedures of milking equipment and suckling equipment on Japanese dairy farms, and the cleanliness of bucket milkers, suckling buckets, milk receivers, and bulk tanks, using adenosine triphosphate (ATP) bioluminescence test. Bulk tanks (except one bulk tank) and milk receivers were washed by automated cleaning, but all bucket milkers and suckling buckets were washed by manual cleaning. Detergents were often not used to clean bucket milkers and suckling buckets. The log10 transformed relative luminescence units (LRLU) of equipment washed by manual cleaning was higher than equipment washed by automated cleaning. Clean surfaces (≤2.2 LRLU) were only observed on the bulk tank and the milk receiver. More than 50% of the LRLU of the mouthpiece, the rubber packing of claw, and the nipple of the suckling bucket were determined dirty. These results suggest that the cleanliness of the bucket milkers and the suckling buckets washed by manual cleaning was lower than that of the equipment washed by automated cleaning, and may be due to insufficient cleaning procedures.

Comparative evaluation of the cost and efficiency of four types of sexing methods for the production of dairy female calves.

This study was conducted to evaluate and compare the economic benefits of different embryo sexing methods, based on the cost per female dairy calf produced. Female calves were produced from four kinds of female embryos: (1) those collected from superstimulated donors at 7-8 days after artificial insemination (AI) with X-sorted semen; (2) those sex-determined by loop-mediated isothermal amplification assay of a biopsy sample of embryos collected from superstimulated donors after AI with conventional unsorted semen; (3) those obtained by invitro embryo production (IVEP), using X-sorted semen and in vitro-matured oocytes collected from donors by ovum pick-up (OPU); and (4) those obtained by IVEP, using X-sorted semen and oocytes collected by OPU after dominant follicle ablation and follicle growth stimulation of the donors. The respective productivities of female calves per technical service and the total production cost per female calf of each sexing method were compared. The production cost per female calf (66,537 JPY), as calculated from the number of female calves per service (1.30), pregnancy rate of transfer (42.9%), rate of female calves obtained (92.9%), and total cost of the method (56,643 JPY plus embryo transfer fee), was less for IVEP with X-sorted semen and follicular growth-stimulated (FGS) oocytes than for the other groups (P < 0.05). The results demonstrate that embryo production with X-sorted semen and FGS oocytes provides a more efficient method for producing female calves than the other embryo sexing methods.

Effects of supplementing an active dry yeast product on rumen microbial community composition and on subsequent rumen fermentation of lactating cows in the mid-to-late lactation period.

The effects of supplementing feed of cows in mid-to-late lactation with an active yeast product (Actisaf Sc 47) were evaluated using 15 Holstein cows in a replicated 3 × 3 Latin square design. The animals were fed a mixed ration with 33% neutral detergent fiber, consisting of timothy hay (29.8%), a commercial concentrate (70.0%) and commercial calcium triphosphate (0.2%), twice daily to meet 105% of their energy requirement. Yeast supplement was set at 0, 5 and 10 g per day over 21-day periods, each of which consisted of 14 days for adaptation followed by 7 days of data collection. Milking performance, plasma metabolite parameters, rumen volatile fatty acids, lipopolysaccharide and microbial properties were measured. Although there were no significant differences in feeding and milking performance or blood parameters associated with supplementation, the acetate to propionate ratio in the rumen fluid tended to decrease (P = 0.08). The population of Bacteroidetes tended to be less prominent (P = 0.07) and the fibrolytic bacterium Fibrobacter significantly increased (P < 0.05) in the rumen fluid of the yeast 10 g group compared with that of the control. These data suggest that effects of supplementing live yeast to cows in mid-to-late lactation may be limited to microbial composition and fermentation characteristics in the rumen.

Differences in rumen fermentation characteristics between low-yield and high-yield dairy cows in early lactation.

Relationship between rumen fermentation parameters, blood biochemical profiles and milk production traits in different yielding dairy cows during early lactation was investigated. Twelve dairy cows were divided into two groups based on their milk yield, that is low-yield (LY) and high-yield (HY) groups. Rumen fluid and blood were collected at 3 weeks prepartum and 4, 8 and 12 weeks postpartum. Results showed that proportions of acetate, propionate to total short chain fatty acids and acetate : propionate ratio were changed (P < 0.05) in both groups during the peripartum period, whereas butyrate and acetate : butyrate ratio were only altered in the HY group. Blood cholesterol, beta-hydroxybutyric acid (BHBA) and glutamic oxaloacetic transaminase in the HY group were higher (P < 0.01) than those in the LY group. Principal component analysis revealed that milk yield and milk compositions were differently clustered between groups. These parameters showed similar direction with dry matter intake in the HY group and adverse direction in the LY group. Linear regression analysis indicated that butyrate was positively correlated with BHBA (P < 0.05) in the HY group. This study suggests that cows in the HY group seem to accommodate appropriately to negative energy balance in early lactation through rumen fermentation.

Dynamics of lingual antimicrobial peptide, lactoferrin concentrations and lactoperoxidase activity in the milk of cows treated for clinical mastitis.

The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day -6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days -6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 (<5 × 10(6) cell/mL) were significantly higher and lower than those in cows with a higher SCC, respectively. These results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factor in milk of cows treated for clinical mastitis.

Improvement of preimplantation development of in vitro-fertilized bovine zygotes by glucose supplementation to a chemically defined medium.

The influences of glucose supplementation on early development of bovine embryos in BSA-free synthetic oviduct fluid were examined. Among the groups supplemented with 1.5, 2.0, 4.0 or 5.6 mM glucose either at 0, 72 or 144 hr after fertilization, blastocysts yield significantly increased in the group supplemented with 4.0 mM glucose 144 hr after fertilization compared to the controls without glucose supplementation. The results suggest that appropriate amounts of glucose supplemented to the medium at the specific stage of embryo culture may be useful for the production of bovine blastocysts.

Relationship between stress levels of the antepartum cow and her newborn calf.

We investigated the relationship of the stress levels of the dam before and after delivery to that of her offspring soon after birth. Eight pregnant cows were penned 7 days before calving. Blood was taken from the jugular vein of cows at -7, 1, 2 and 3 days from calving. Blood was also taken from newborn calves at 6 h and 1 and 2 days after birth. Concentrations of cortisol and immunoglobulin G in blood and colostrum were examined. Pearson's correlation coefficient showed that the higher the plasma cortisol concentration of a cow before calving, the higher that of its calf after birth (all P < 0.01). In addition, path analysis demonstrated that the direct effect of the plasma cortisol concentration of the dam before calving on the plasma cortisol concentration of her calf after birth was 0.971 (P < 0.01). However, the colostrum cortisol concentration correlated with neither plasma cortisol concentrations of cows before calving nor that of calves after birth. Unlike cortisol, a clear correlation of immunoglobulin G concentrations in plasma and colostrum was not observed between cows and calves. The results indicate stress is transferred from a cow to her newborn calf not by way of the colostrum but through the placenta.

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows.

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.

Effects of growth and dietary crude protein level until first insemination on milk production during first lactation in Holstein heifers.

To decrease the age at first calving in Holsteins, the effects of average daily body weight gain (ADG) and crude protein (CP) level until first insemination on growth performance and milk production were examined. The MM group had a target ADG of 0.75 kg and received a diet with a CP level of 14%. The HM and HH groups had a target ADG of 1 kg; both these groups received a diet with CP levels 14% and 16%, respectively. The ADG in the HM and HH groups was 1.1 kg, whereas in the MM group it was 0.97 kg (P < 0.01). The HM and HH groups showed no differences in withers height at body weight 350 kg. The ages at first calving in MM, HM and HH groups were 23.1, 21.0 and 21.8 months, respectively. The HM and HH groups had lower milk yield at day 305 than the MM group (P < 0.01). These results suggest that growth performance until first insemination should be maintained at an ADG of 0.97 kg or less with a CP level of approximately 14%, to shorten time until first insemination and prevent the decrease of milk yield.

Effect of cellooligosaccharide or synbiotic feeding on growth performance, fecal condition and hormone concentrations in Holstein calves.

We investigated the effect of cellooligosaccharide (CE) or a combination of dextran and Lactobacillus casei ssp. casei strain JCM1134(T) (synbiotic; SB) feeding on growth performance, fecal condition and hormone concentrations in Holstein calves. Fifty-two female Holstein calves were randomly assigned to three treatment groups: CE feeding group (n = 16), SB feeding group (n = 18), and control group (n = 18). Body weight at 90 days of age, as well as daily body weight gain (DG) and feed efficiency after weaning to 90 days of age were greater (P < 0.05) in the CE feeding group than in the control group. The total fecal score tended to be lower (P < 0.1) in the SB feeding group than in the control group. Plasma insulin concentration was higher (P < 0.05) in the CE feeding group than in the control group at 90 days of age. Our results indicate that CE feeding improved DG and feed efficiency in calves. On the other hand, there was less effect on growth performance and fecal Escherichia coli counts in calves fed SB.

Calf production from vitrified bovine sexed embryos following in-straw dilution.

The objective of this study was to develop an in-straw dilution method suitable for direct transfer of vitrified bovine sexed embryos. Embryo sexing was performed by molecular diagnosis. Several sexed and vitrified-warmed embryos were transferred after evaluation of morphologically embryonic survival at warming and in-straw dilution (Evaluation group). The other embryos were immediately directly transferred to recipients without first being expelled from the straws after in-straw dilution (Non-evaluation group). The pregnancy rates of vitrified sexed embryos were 38.7% and 34.8% in the Evaluation group and Non-evaluation group, respectively, which were not significantly different. The viability of lower quality embryos before vitrification tended to be lower (P = 0.087) than that of the higher quality embryos regardless of evaluating embryos after warming and in-straw dilution. The abortion rates were similar, and there was no difference between the two groups (13.9% and 12.5%, respectively). These results demonstrate that vitrified bovine sexed embryos can be vitrified and diluted by the in-straw method and that the vitrified and warmed sexed embryos can develop to term.

Viability of porcine embryos after vitrification using water-soluble pullulan films.

The efficiency of a porcine embryo vitrification method that uses water-soluble films of pullulan, a naturally-occurring polysaccharide polymer, was compared with two other types of vitrification methods using different devices and solutions for vitrification and warming. Blastocysts collected in vivo and vitrified by the conventional straw (ST), Cryotop((R)) (MVC) or pullulan film vitrification (PFV) methods were stored in liquid nitrogen for a certain period of time, after which the cryoprotective agents were removed by stepwise dilution. Fresh embryos were used as controls for the non-vitrification group. The vitrified-warmed embryos were incubated in TCM199 with 0.1 mM beta-mercaptoethanol and 20% fetal bovine serum for 24 h at 38.5 C in humidified air with 5% CO(2) to evaluate their viability. The survival rate of embryos in the ST group (48.3%) was significantly lower than that of those in the MVC (70.7%), PFV (79.0%) and non-vitrification (94.4%) groups. The oxygen consumption rate after vitrification was significantly lower than that before vitrification in the ST group, but was not significantly different in the MVC and PFV groups. Both the oxygen consumption rates of embryos after warming and the live cell numbers in the ST group were lower than those in the MVC group, while they did not differ significantly between the PFV and MVC groups. There was a correlation between the oxygen consumption rate and the number of live cells in vitrified embryos after warming. Our results demonstrated that in vivo-derived porcine embryos could be vitrified using pullulan films.

Classification of morphological changes based on the number of cleavage divisions in bovine embryos.

Quantification based on cleavage division (CD) of bovine preimplantation embryos facilitates quantitative analyses of embryonic developmental processes because CD occurs roughly once each day for all blastomeres for up to at least 9 days after ovulation. Therefore, embryonic morphological changes during this period were classified according to CD number. In this study, embryos collected from superovulated donors 0-9 days after ovulation were first classified morphologically into 14 conventional developmental stages. The total cell numbers (TCN) of embryos were measured using the air-dry method. The respective CD numbers of the embryos were then determined using logarithmic transformation of the TCN. The CD numbers of embryos were increased 0-10th with 11 stages. The 0th CD corresponded to 1-cell stage embryos; the 1st CD corresponded to 2-cell stage embryos; the 2nd CD corresponded to 3-4-cell stage embryos; the 3rd CD corresponded to 5-8-cell stage embryos; the 4th CD corresponded to 9-16-cell stage embryos, the 5th CD corresponded to morulae (17-32-cell stage embryos); and the 6th CD corresponded to the compact morulae. Furthermore, the 7th CD included early blastocysts to blastocysts. The 8th CD included expanded, collapsed and hatching blastocysts. The 9th CD included hatched blastocysts. The 10th CD included expanding-hatched blastocysts. The relationship between the CD number and the morphological characteristics of the bovine embryos 0-9 days after ovulation was expressed using a linear equation, and this revealed a high degree of correlation (y=0.98x-0.96, r=0.99). These results suggest that morphological changes of bovine embryos can be classified accurately using an 11-stage classification system based on the number of cleavages.

Transition of cell numbers in bovine preimplantation embryos: in vivo collected and in vitro produced embryos.

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.

Inhibition of tetrandrine on epidermal growth factor-induced cell transformation and its signal transduction.

BACKGROUND: The mouse epidermal JB6 cell system is a model for studying tumor promotion. We used the JB6 Cl 41 cell line to examine the mechanism of the anti-tumor-promoting effect of tetrandrine, an alkaloid isolated from Stephania tetrandra S Moore. MATERIALS AND METHODS: The anti-tumor-promoting effect of tetrandrine was evaluated by assay of inhibition of epidermal growth factor (EGF)-induced transformation of JB6 Cl 41 cells in soft agar. The activity of activator protein-1 (AP-1), a transcription factor, was analyzed using the AP-1-dependent reporter assay. Phosphorylation of extracellular-signal regulated kinases (ERKs) and Akt, a pivotal effector of phosphatidylinositol 3-kinase (P13K), was detected by Western blotting. RESULTS: Tetrandrine significantly blocked EGF-induced cell transformation, attenuated EGF-induced AP-1 activation, and inhibited phosphorylation of ERKs, which regulates AP-1 activation. It also tended to suppress EGF-induced Akt phosphorylation. CONCLUSION: Our results indicate that tetrandrine inhibits EGF-induced transformation of JB6 cells by blocking the activation of ERKs, AP-1 and Akt.

Antitumor effect of geranylamine derivatives on human hepatoma.

In this study, we examined the antitumor activities of isoprenoid derivatives conjugated with substrates of energy metabolism in human hepatoma-bearing athymic mice. Among these compounds, N-geranylpyruvic amide, N-geranyl-p-pyruvaminobenzoic amide, N,N'-digeranylmalic diamide and N,N'-digeranyl-O-acetylmalic diamide had strong antitumor effects. These geranylamine derivatives also inhibited in vitro cell growth. Sugar conjugates of geranylamine, geranic acid and mevalonic acid did not show any antitumor effect in vivo or in vitro. Although the geranylamine derivatives had no impact on the cell cycle distribution at 24 h, a sub-G1 (apoptotic) peak of varying magnitude was seen in DNA histograms of cells treated with the derivatives for 48 h. However, the geranylamine derivatives did not inhibit protein isoprenylation, which has been reported in cancer cells treated with several natural isoprenoids. These results suggest that the geranylamine derivatives conjugated with malic acid and pyruvic acid have a different mechanism of antitumor activity from that of natural isoprenoids.