Mechanical prophylaxis is a heparin-independent risk for anti-platelet factor 4/heparin antibody formation after orthopedic surgery.

Platelet-activating antibodies, which recognize platelet factor 4 (PF4)/heparin complexes, induce spontaneous heparin-induced thrombocytopenia (HIT) syndrome or fondaparinux-associated HIT without exposure to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). This condition mostly occurs after major orthopedic surgery, implying that surgery itself could trigger this immune response, although the mechanism is unclear. To investigate how surgery may do so, we performed a multicenter, prospective study of 2069 patients who underwent total knee arthroplasty (TKA) or hip arthroplasty. Approximately half of the patients received postoperative thromboprophylaxis with UFH, LMWH, or fondaparinux. The other half received only mechanical thromboprophylaxis, including dynamic (intermittent plantar or pneumatic compression device), static (graduated compression stockings [GCSs]), or both. We measured anti-PF4/heparin immunoglobulins G, A, and M before and 10 days after surgery using an immunoassay. Multivariate analysis revealed that dynamic mechanical thromboprophylaxis (DMT) was an independent risk factor for seroconversion (odds ratio [OR], 2.01; 95% confidence interval [CI], 1.34-3.02; P = .001), which was confirmed with propensity-score matching (OR, 1.99; 95% CI, 1.17-3.37; P = .018). For TKA, the seroconversion rates in patients treated with DMT but no anticoagulation and in patients treated with UFH or LMWH without DMT were similar, but significantly higher than in patients treated with only GCSs. The proportion of patients with ≥1.4 optical density units appeared to be higher among those treated with any anticoagulant plus DMT than among those not treated with DMT. Our study suggests that DMT increases risk of an anti-PF4/heparin immune response, even without heparin exposure. This trial was registered to www.umin.ac.jp/ctr as #UMIN000001366.

Cryoprotective effect of antifreeze polyamino-acid (Carboxylated Poly-l-Lysine) on bovine sperm: A technical note.

In this study, investigating Carboxylated Poly-l-Lysine (CPLL) for its effectiveness as a new cryoprotectant for bovine sperm is aimed. CPLL is an ampholytic polymer compound, has cryoprotective properties similar to those of anti-freeze protein. The cryopreservation medium used for control group consisted of 6.5% (v/v) glycerol, the cryopreservation medium used for experimental group consisted of 3.25% (v/v) glycerol + 0.5% (w/v) CPLL. There was no consequential difference in sperm motility parameter after thawing whereas there was huge distinction for sperm membrane integrity rate (control vs experimental; 49.6 vs 60.7%, P < 0.01). Conception rate of artificial insemination of experimental group was significantly higher than that of control group (79.0% vs 53.1%, P < 0.01). These results suggest CPLL has protected sperm membrane and leads to improve fertility. This is the first report using CPLL for bovine sperm cryopreservation, it is also expected CPLL can be applied to other animal species.

Cryopreservation of bovine somatic cells using antifreeze polyamino-acid (carboxylated poly-L-lysine).

Carboxylated poly-L-lysine (CPLL) is an ampholytic polymer compound, obtained by converting 65 mol% of amino groups to carboxyl groups after synthesizing ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective properties similar to those of anti-freeze protein. The addition of CPLL to freezing medium has been reported to improve the post-thawing survival rate of murine cells, human induced pluripotent stem (iPS) cells, embryonic stem (ES) cells and embryos. In this study, investigating CPLL for its effectiveness as a new cryoprotective material is aimed. In experiments with bovine somatic cells, CPLL was suggested to have an equal or superior cryoprotective effect to dimethyl sulfoxide (DMSO), the conventional material for cellular frozen storage, based on the results for post-thawing cell survival and proliferation rates. CPLL was demonstrated to have another advantage; thawed cells can be cultured without removing the cryopreservation medium when CPLL is used, but not when DMSO is used. These results suggest that CPLL could be used as cryoprotective material for bovine cells. It is also expected that CPLL can be applied to embryo and oocytes storage for cattle, and similar functions for cells and embryos of other animal species.

Aberrant patterns of X chromosome inactivation in bovine clones.

In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.

LL5beta directs the translocation of filamin A and SHIP2 to sites of phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) accumulation, and PtdIns(3,4,5)P3 localization is mutually modified by co-recruited SHIP2.

Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P(3) regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P(3) and the reciprocal regulation of PtdIns(3,4,5)P(3) itself. LL5beta, whose subcellular localization is directed by membrane PtdIns(3,4,5)P(3), recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P(3) accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5beta by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5beta or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5beta without its PtdIns(3,4,5)P(3)-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P(3) accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P(3) at the same location.

Filamin A-interacting protein (FILIP) regulates cortical cell migration out of the ventricular zone.

Precisely regulated radial migration out of the ventricular zone is essential for corticogenesis. Here, we identify a mechanism that can tether ventricular zone cells in situ. FILIP interacts with Filamin A, an indispensable actin-binding protein that is required for cell motility, and induces its degradation in COS-7 cells. Degradation of Filamin A is identified in the cortical ventricular zone, where filip mRNA is localized. Furthermore, most ventricular zone cells that overexpress FILIP fail to migrate in explants. These results demonstrate that FILIP functions through a Filamin A F-actin axis to control the start of neocortical cell migration from the ventricular zone.

Assisted hatching of poor-quality bovine embryos increases pregnancy success rate after embryo transfer.

Embryos of good, fair and poor quality were collected from superovulated cows and subjected to zona cutting (ZC) treatment using a needle under either an inverted microscope or a stereomicroscope. One (single transfer) or 2 (twin transfer) embryos with or without prior ZC treatment were transferred nonsurgically to recipients. Without the ZC treatment, lower embryonic quality resulted in lower pregnancy success rates. However, the ZC treatment increased the pregnancy success rate following transfer of poor-quality embryos, but not the pregnancy rate after transfer of good- or fair-quality embryos. No differences were observed between the pregnancy success rates after the transfer of embryos treated under the inverted microscope and those after transfer of embryos treated under the stereomicroscope, and this was the same after single and twin transfer. Moreover, ZC treatment of embryos prior to transfer did not result in an increased abortion rate, irrespective of the number of transferred embryos. In conclusion, ZC treatment improves pregnancy success rates following transfer of poor-quality embryos. Moreover, the results indicate that ZC treatment by using a stereomicroscope is practical for on-farm application.

Bovine serum miR-21 expression affected by mastitis.

The expression levels of circulating microRNAs (miRNAs) can be affected by disease. The miRNA released from cells within exosomes can act as a remote communication tool and can participate in inflammatory response regulation. Therefore, circulating miRNA has the potential to be an indicator of local disease. The objective of this study was to investigate the serum level of bovine mastitis-related miRNAs. We found that miR-16 expression in serum was affected by hemolysis. The expression levels of miR-21 in serum were increased significantly in cows with mastitis compared with unaffected controls; however, the expression levels of miR-146a, miR-155, miR-222 and miR-383 in cows with mastitis were unchanged. We further verified the upregulation of miR-21 in the serum of cows with mastitis using a digital PCR system. Although the sensitivity and specificity of miR-21 in the serum to detect bovine mastitis was inferior to miRNA biomarkers in the milk, the significant increase of miR-21 in serum may reflect the impact of local inflammation on the systemic reaction.

Risk assessment of meat and milk from cloned animals.

Research on, and commercialization of, cloned cattle has been conducted for more than 20 years. Early techniques relied on the physical splitting of embryos or using embryo cells for nuclear transfer to generate cloned animals. Milk and meat from these animals entered into the human food market with no evidence of problems. With the advent of nuclear transfer, which enables the direct transference and preservation of high-value meat- and milk-producing genotypes to offspring, concerns have been raised about whether the products from somatic cell nuclear transfer-produced animals are safe for human consumption. Studies on the biochemical properties of food products from cloned and noncloned animals have thus far not detected any differences. All data to date indicate no significant differences in the measured parameters between animals created by nuclear transfer and normally bred animals. Public acceptance of cloned animal products depends upon forthcoming US Food and Drug Administration approval along with convincing safety data.

Bovine milk transcriptome analysis reveals microRNAs and RNU2 involved in mastitis.

Mastitis is a common inflammatory infectious disease in dairy cows. To understand the microRNA (miRNA) expression profile changes during bovine mastitis, we undertook a genome-wide miRNA study of normal milk and milk that tested positive on the California mastitis test for bovine mastitis (CMT+). Twenty-five miRNAs were differentially expressed (23 miRNAs upregulated and two downregulated) during bovine mastitis relative to their expression in normal milk. Upregulated mature miR-1246 probably derived from a U2 small nuclear RNA rather than an miR-1246 precursor. The significantly upregulated miRNA precursors and RNU2 were significantly enriched on bovine chromosome 19, which is homologous to human chromosome 17. A gene ontology analysis of the putative mRNA targets of the significantly upregulated miRNAs showed that these miRNAs were involved in binding target mRNA transcripts and regulating target gene expression, and a Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the upregulated miRNAs were predominantly related to cancer and immune system pathways. Three novel miRNAs were associated with bovine mastitis and were relatively highly expressed in milk. We confirmed that one of the novel mastitis-related miRNAs was significantly upregulated using a digital PCR system. The differentially expressed miRNAs were involved in human cancers, infections, and immune-related diseases. The genome-wide analysis of miRNA profiles in this study provides insight into bovine mastitis and inflammatory diseases. DATABASES: The miRNAseq generated for this study can be found in the Sequence Read Archive (SRA) under BioProject Number PRJNA421075 and SRA Study Number SRP126134 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA421075).

Genetic heterogeneity among strains of Treponema phagedenis-like spirochetes isolated from dairy cattle with papillomatous digital dermatitis in Japan.

Papillomatous digital dermatitis (PDD) is an infectious foot disease of cattle that is prevalent throughout the world. Although it has been prevalent in Japan since the first case was reported in 1992, full epidemiological and bacteriological examinations have not been conducted. We collected 91 lesions of PDD from 80 dairy cattle on 12 farms in eight regions of Japan to isolate the spirochetes that are frequently detected in lesions. We isolated 40 strains of spirochetes from 24 cattle (30.0%) by a simple two-step culture technique, in which the biopsy samples were incubated at 4 degrees C for 48 to 72 h in an enrichment broth supplemented with antibiotics, which improved the rate of isolation, and then inoculated on selective agar plates. All spirochetes examined were catalase positive and oxidase negative and showed weak beta-hemolytic activity. Enzyme activities were identical to those of Treponema phagedenis ATCC 27087. Sequencing of the 16S rRNA gene showed that all strains isolated had >99% identity to those of the T. phagedenis type strain and of T. phagedenis-like strains isolated from PDD lesions in the United States and Europe. Pulsed-field gel electrophoresis and PCR-based random amplified polymorphism DNA methods revealed considerable diversity among strains isolated not only from different cattle but also from the same individuals. These findings may provide further evidence for the role of these treponemes in the pathogenesis of persistent PDD.

Effect of combined vaccination for Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni to prevent respiratory diseases in young Japanese Black calves in the field.

To evaluate the preventive effects of combined vaccination for Pasteurella multocida, Mannheimia haemolytica and Histophilus somni on respiratory diseases in Japanese Black calves, 295 calves at one farm were alternately assigned to two groups; 147 calves received the vaccine at 4 and 8 weeks of age (vaccination group), and the other 148 calves did not receive vaccine (control group). The incidences of respiratory diseases were 25.9 and 70.9% in the vaccination and control groups, respectively, and the odds ratio for comparison between the two groups was 0.143 (95% confidence interval: 0.086-0.238). Administration of the multiple vaccine to Japanese black calves might be one of effective factor for prevention of respiratory diseases.

Evaluation of the effect of a 3rd GnRH injection administered six days after the 2nd GnRH injection of Ovsynch on the reproductive performance of Japanese black cows.

This study was designed to evaluate the reproductive performance of Japanese black cows following the 3rd injection of gonadotropin releasing hormone (GnRH) analogue administered concurrently with Ovsynch-based treatment on day 6 (day 1 = the day of ovulation). In Experiment 1, 12 cows were allocated into three groups: a control group that was subjected to Ovsynch treatment and then injected with a placebo on day 6; group 1 (Ovsynch + GnRH), which was subjected to Ovsynch treatment and was injected with GnRH analogue on day 6, and group 2 (Ovsynch + controlled internal drug-release (CIDR) + GnRH), which received Ovsynch-CIDR treatment and was injected with GnRH analogue on day 6. Blood collection and ultrasonographic observation of the ovaries were conducted daily. Both treatments induced the formation of an accessory corpus luteum and significantly increased the cross-sectional area of the luteal tissue when compared to the control. However, plasma progesterone (P4) was significantly higher in the treatment groups than in the control group on days 11, 12, 17 and 18 in the group 1 and from day 10 to 21 in the group 2. In Experiment 2, 41 cows were assigned to the same three groups described above and then artificially inseminated on day 1. The pregnancy rates on day 45 did not differ among groups. In conclusion, administration of GnRH analogue on day 6 following Ovsynch-based treatment did not improve the reproductive performance of Japanese black cows, even though the P4 concentration was higher in groups that received the GnRH.

Luteal lifespan and fertility after estrus synchronization in goats.

The present experiment aims to examine the efficiency of estrus synchronization using progesterone and equine chorionic gonadotrophin (eCG) and to look at luteal function. During the non-breeding and breeding season, 5 adult female Korean native goats were injected intramuscularly with 2.5 ml of physiological saline as the control. A progesterone impregnated intravaginal sponge was then kept in the same goats for 10 days followed, after a week, by an intramuscular injection of 500 IU eCG. Five adult female Nubian goats were mated with a fertile buck during the non-breeding season. During the non-breeding season 2 of the 5 goats showed a normal estrous cycle (ranging from 18 to 21 days) and 3 a short estrous cycle (ranging from 3 to 6 days). During the breeding season the equivalent figures were 1 and 2. The major axes of the corpus luteum (CL) were measured by means of calipers built into the ultrasonography system, and the concentrations of plasma progesterone (P(4)) were determined by double antibody radioimmunoassay. The mean major axes of the CL in goats showing the short cycle (6.1 +/- 0.5 mm) was significantly smaller than in those showing the normal cycle (8.9 +/- 0.5 mm; p < 0.01) and also the value of P(4) in goats showing the short cycle (4.2 +/- 2.1 ng/ml) was significantly lower than for those showing the normal cycle (10.3 +/- 4.3 ng/ml; p < 0.05) at day 3 following ovulation. Three out of 5 Nubian goats became pregnant but only one goat carried to full term. The present experiment indicated that a combination of progesterone and eCG was effective in inducing estrus, although it resulted in a high incidence of short luteal lifespan. The low kidding rate and high incidence of embryonic loss may be due to the instability of the luteal lifespan.

Effect of Beta-Carotene Supplementation on the Serum Oxidative Stress Biomarker and Antibody Titer against Live Bovine Respiratory Syncytial Virus Vaccination in Japanese Black Calves.

The purpose of this study was to determine the effect of vaccination and beta-carotene supplementation on blood oxidative stress and antibody response in calves. Thirty Japanese Black calves were randomly assigned to two groups. Fifteen calves received 20 mg of beta-carotene supplemented into their daily provided rations from 2 to 8 weeks of age (BC group), and the other 15 calves did not receive the daily beta-carotene supplement (control group). All calves received a commercially available modified live bovine respiratory syncytial (RS) virus vaccine at 4 and 8 weeks of age. Blood samples were taken at 2, 4, 8, and 12 weeks of age. At 4 weeks of age, the concentration of reactive oxygen metabolites within serum were significantly lower in the BC group than the concentrations measured in the control group. Also at 4 weeks of age, the concentration of biological antioxidant capacity within serum was significantly higher in the BC group than the concentrations measured in the control group. Both groups showed a gradual decrease of antibody titers to live bovine RS virus in the samples taken from 2 to 12 weeks of age. These results confirmed that beta-carotene supplementation decreased oxidative stress. However, beta-carotene supplementation did not affect the antibody response to live bovine RS virus vaccination, perhaps due to the presence of the maternal antibody.

Serial bull cloning by somatic cell nuclear transfer.

Although the list of species successfully cloned continues to grow, serial cloning has not been reported in species other than the mouse. Here we describe two live births of second-generation clones of a bull. Clones of the first and second generations appear healthy and have normal telomere lengths. Our attempts to produce the third generation of clones were unsuccessful.

Hydromyelia in a Japanese Black calf.

A Japanese Black calf, manifested by clinical signs of inability to stand and extending hind limbs after birth, was investigated clinically and pathologically. In the neurological inspection, gastrocnemius reflex and patellar tendon reflex of the right hind limb were hyperactive, and gastrocnemius reflex and crossed extension reflex of the left hind limb were also hyperactive. Magnetic resonance imaging (MRI) examination showed a cavity in the cord at the area of the third and fourth lumbar vertebrae in T2-weighted imaging. After the calf was euthanized, necropsy confirmed the swelling of the spinal cord at the third and fourth lumbar vertebrae. The histopathological examination confirmed that the central canal remained open, and the inner surface of central canal was lined with ependymal cells. Therefore, this calf was diagnosed to have hydromyelia of the lumbar cord. This study indicated that MRI was useful for diagnosing myelodysplasia such as hydoromyelia. This is the first report of hydoromyelia in Japanese Black calf.

Inflammation-related microRNA expression level in the bovine milk is affected by mastitis.

MicroRNA (miRNA) in tissue and liquid samples have been shown to be associated with many diseases including inflammation. We aimed to identify inflammation-related miRNA expression level in the bovine mastitis milk. Expression level of inflammation-related miRNA in milk from mastitis-affected and normal cows was analyzed using qPCR. We found that expression level of miR-21, miR-146a, miR-155, miR-222, and miR-383 was significantly upregulated in California mastitis test positive (CMT+) milk. We further analyzed these miRNA using a chip-based QuantStudio Digital PCR System. The digital PCR results correlated with those of qPCR, demonstrating upregulation of miR-21, miR-146a, miR-155, miR-222, and miR-383 in CMT+ milk. In conclusion, we identified miRNA that are upregulated in CMT+ milk. These miRNA exhibited sensitivity and specificity greater than 80% for differentiating between CMT+ milk and normal milk. Our findings suggest that inflammation-related miRNA expression level in the bovine milk was affected by mastitis, and miRNA in milk have potential for use as biomarkers of bovine mastitis.

Interpreting gelatinase activity in tumor tissue and serum as a prognostic marker of naturally developing canine tumors.

To evaluate the clinical usefulness of tissue and serum gelatinase activity as a prognostic marker of canine tumors, tissue samples from 60 tumors and corresponding serum samples from the same animals were collected at the time of biopsy and surgery. On the basis of histopathology and clinical aggressiveness of metastasis and recurrence (MR), the cases were divided into 6 categories: non-inflammatory (Inf(-)) and inflammatory (Inf(+)) benign, and the Inf(-) MR(-), Inf(-) MR(+), Inf(+) MR(-), and Inf(+) MR(+) malignant. Gelatinase activity was determined semi-quantitatively using gelatin zymogram with a gelatinase standard from cultured canine peripheral blood mononuclear cells stimulated with lipopolysaccharide. No significant difference in gelatinase activities in tissue extracts was evident between the benign and malignant tumors. Inf(+) benign tumors, as well as Inf(-) MR(+), Inf(+) MR(-) and Inf(+) MR(+) malignant tumors, showed significantly higher tissue gelatinase activity than Inf(-) benign. The tissue activity in Inf(-) MR(-) malignant was significantly lower than in Inf(+) MR(-) and Inf(+) MR(+) malignant. The serum activity was significantly higher in the malignant cases than in the control and the benign. Inf(-) MR(+), Inf(+) MR(-) and Inf(+) MR(+) malignant tumors induced significantly higher gelatinase activity in serum than Inf(-) benign tumors. Gelatinase activity in serum was positively correlated with that in tumor extracts. Increased gelatinase in tumor tissue and serum may be correlated with inflammation as well as tumor aggressiveness, and thus should be used in combination with histopathology for predicting tumor metastasis or recurrence.

Diagnostic imaging of intra-abdominal cyst in heifer using the computed tomography.

A 10-month-old Japanese black heifer was diagnosed as having an intra-abdominal cyst using computed tomography (CT). Through a posterior ventral midline incision, the cyst was removed, and the heifer completely recovered after the surgery. CT scans enabled detection of the intra-abdominal cyst and measurements of the diameter of the cyst before the surgery.