Evaluation of bovine embryo quality based on gene expression profiling using whole-transcriptome amplification.

This study aimed to develop a method to evaluate the quality of bovine in vitro fertilized (IVF) embryos based on gene expression profiling via whole-transcriptome amplification. The expression of 11 developmentally important genes in individual bovine in vivo-derived (IVD) and IVF embryos were examined. Gene expression profiling was conducted by classifying the expression level of each gene in individual embryos as low, medium, or high. The IVF group had a higher (P < 0.01) proportion of embryos with low expression of SOX2, NANOG, and FGF4. In addition, a correlation analysis between the expression levels of each gene in individual embryos demonstrated that the relationship between gene expression differed with respect to IVD and IVF embryos. Our results suggest that the expression profiling of developmentally important genes using IVD embryos as normal controls could be a useful indicator for evaluating the quality of bovine IVF embryos.

Serum bta-miRNA-375 as a potential biomarker for the early diagnosis of enzootic bovine leukosis.

To identify a biomarker for the early diagnosis of enzootic bovine leukosis (EBL) caused by bovine leukemia virus (BLV), we investigated the expression of a microRNA, bta-miR-375, in cattle serum. Using quantitative reverse-transcriptase PCR analysis, we measured bta-miR-375 levels in 27 samples from cattle with EBL (EBL cattle), 45 samples from animals infected with BLV but showing no clinical signs (NS cattle), and 30 samples from cattle uninfected with BLV (BLV negative cattle). In this study, we also compared the kinetics of bta-miR-375 with those of the conventional biomarkers of proviral load (PVL), lactate dehydrogenase (LDH), and thymidine kinase (TK) from the no-clinical-sign phase until EBL onset in three BLV-infected Japanese black (JB) cattle. Bta-miR-375 expression was higher in NS cattle than in BLV negative cattle (P < 0.05) and greater in EBL cattle than in BLV negative and NS cattle (P < 0.0001 for both comparisons). Receiver operating characteristic curves demonstrated that bta-miR-375 levels distinguished EBL cattle from NS cattle with high sensitivity and specificity. In NS cattle, bta-miR-375 expression was increased as early as at 2 months before EBL onset-earlier than the expression of PVL, TK, or LDH isoenzymes 2 and 3. These results suggest that serum miR-375 is a promising biomarker for the early diagnosis of EBL.

Interferon-Stimulated Gene Expression in Peripheral Blood Leucocytes as a Convenient Prediction Marker for Embryo Status in Embryo-Transferred Japanese Black Cows during the Peri-Implantation Period.

Pregnancy diagnosis during early gestation is important for cattle reproduction. The expression of interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) was studied in embryo-transferred (ET) Japanese Black cattle. ISGs in PBLs-ISG15, MX1, MX2, and OAS1-were detected in multiple ovulation ET cattle using a real-time quantitative polymerase chain reaction, and receiver operating characteristic (ROC) curve analysis was performed. Gestational status was predicted using the average ISG levels during the normal estrous cycle (AVE) and the Youden index from the ROC curve analysis as cutoff values. The ISG15, MX1, and MX2 levels were significantly higher in pregnant cattle (n = 10) than in non-pregnant cattle (n = 23) on gestation day 21, whereas the levels of all ISGs were similar between non-pregnant and non-pregnant cattle with late embryonic death (n = 7). ISG15, MX1, and MX2 appropriately predicted the gestational status of ET cows. The statistical evaluation of the diagnostic accuracy in ET cows on day 21 of gestation presented higher values of sensitivity, specificity, accuracy, and positive predictive values of ISG15, MX1, and MX2 using the Youden index than using the AVE. Therefore, ISG15, MX1, and MX2 are excellent biomarkers of gestational status during the peri-implantation period in ET cattle.

Increased T-cell responses that control bovine leukemia virus proviral load in beef cattle under dietary vitamin A restriction for marbling.

Bovine leukemia virus (BLV) proviral load is controlled by T-cell responses, which require vitamin A (VA) derived from food. However, whether dietary VA restriction for marbling impairs the T-cell responses that control BLV proviral load in beef cattle is unknown. We assessed T-cell subsets, interferon (IFN)-γ gene expression, and BLV proviral load in naturally BLV-infected Japanese Black cattle that were fed a diet with decreased VA levels. We found that the percentage of CD4+ T cells increased over time during dietary VA restriction. In addition, BLV proviral load was negatively correlated with the percentage of CD4+ T cells and with the level of IFN-γ gene expression. These observations suggest that dietary VA restriction for marbling enhances T-cell responses that control BLV proviral load and thus does not promote leukemogenesis in fattening beef cattle.

Detection of urinary luteinizing hormone in Japanese black cows after administration of gonadotropin-releasing hormone.

The blood luteinizing hormone (LH) surge in cows is well studied. However, little is known about urinary LH in cows. This study examined urinary LH concentrations after administration of gonadotropin-releasing hormone (GnRH) in six Japanese black cows to induce LH secretion from the pituitary gland into the bloodstream. Abrupt rises in plasma and urinary LH were observed after GnRH administration. Plasma and urinary LH peaked at 2 and 5 hr, respectively. A positive correlation was observed between plasma LH concentrations and urinary LH amounts. Ovulation was confirmed in the cows after 48 hr of GnRH administration. These data strongly suggest that urinary LH is derived from plasma LH, which triggers ovulation in cows.

Effectiveness of on-farm continuous flow high-temperature short-time pasteurization for inactivation of bovine leukemia virus in milk.

The effectiveness of on-farm continuous flow high-temperature short-time (HTST) pasteurization (i.e., 72°C for 15 s) for the inactivation of bovine leukemia virus (BLV) in milk was investigated with a sheep bioassay. Four sheep that had been inoculated with completely pasteurized milk containing approximately 3.4 × 107 BLV-infected peripheral blood mononuclear cells (PBMC) and treated by either HTST pasteurization or laboratory-scale low-temperature long-time (LTLT) pasteurization (i.e., 60°C for 30 min), remained negative for BLV for at least 17 weeks after inoculation. In contrast, all sheep inoculated with unpasteurized or inadequately pasteurized milk containing the same number of BLV-infected PBMC were tested positive for BLV and anti-BLV antibodies within 3 weeks after inoculation. These results suggest that on-farm continuous flow HTST pasteurization was equivalent value with inactivated BLV on the LTLT procedure and can effectively inactivate BLV in the milk. Therefore, on-farm HTST pasteurization of the pooled colostrum or milk used in automated feeding systems is likely to protect group-housed preweaned calves from BLV infection, thereby improving animal health on dairy farms.

Use of a prediction method for early pregnancy status utilizing receiver operating characteristic curve analysis of peripheral blood leukocyte interferon-stimulated genes in Japanese-Black cattle.

A prediction method for early pregnancy status (pregnant or non-pregnant) in cattle that can be used within 3 weeks after insemination is desired. Interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) have been examined as prediction molecules for determination of pregnancy status. Relative abundances of ISG15 and MX2 gene transcripts in PBLs were suitable biomarkers for the prediction of pregnancy status when there were assessments of Holstein cattle. In the present study, it was determined whether ISG biomarkers are applicable for predicting gestation in Japanese-Black (JB) cattle and evaluation of the applicability of receiver operating characteristic (ROC) analysis procedures for this purpose. There was assessment of the reliability of using average ISG values in PBLs collected during the estrous cycle (AVE) as a cutoff compared to the Youden index cutoff values. Application of AVE to assessment of pregnancy status in JB cattle indicated there was reliable predictions for pregnancy status when using ISG15 and MX2 values on day 21 after insemination, which coincided with the time of assessment in the previous study with Holstein cattle. The area under the curve values of the ROC curves confirmed the reliability of using ISGs to predict pregnancy from days 18 to 21 after insemination. Comparing AVE with Youden index values, there was confirmation of the accuracy of AVE for predicting gestation. The average mRNA transcript abundance values of ISG15 and MX2 may serve as excellent pregnancy biomarkers for cattle within 3 weeks of insemination.

Vertical transmission of Mycoplasma wenyonii in cattle, supported by analysis of the ribonuclease P RNA gene - Short communication.

The vertical transmission of Mycoplasma (M.) wenyonii was investigated in beef cattle raised on a farm in Japan by analysing the ribonuclease P RNA (rnpB) gene sequence using PCR. Peripheral blood samples from 17 dams infected with M. wenyonii and from their neonatal calves were collected and colostrum samples were taken from cows immediately after parturition, and subsequently the blood samples of calves were monitored continuously for three months. At birth on day 0, although no rnpB gene was detected in the colostrum of any of the dams, four (23.5%) of the 17 calves born were positive. At three months after delivery, the number of positive calves decreased to three. Although horizontal transmission by blood-feeding arthropod vectors has been basically accepted as the most common route of haemoplasma infection, these findings suggest that vertical transmission is, at least in part, another most likely route of M. wenyonii infection in cattle.

Diurnal variation of plasma bone markers in Japanese black calves.

To evaluate diurnal variation of plasma bone markers, blood samples were collected from five calves at 2-hr intervals throughout a 24-hr period. Tartrate-resistant acid phosphatase isoform 5b (TRAP5b), carboxy-terminal collagen crosslinks of type-I collagen (CTX), hydroxyproline, bone specific alkaline phosphatase (BALP) and osteocalcin were measured. Cosinor analysis showed a significant rhythm in all bone markers. The acrophase of each bone marker appeared from the early to late morning. The percentage ratio of the amplitude to mesor and the within-subject variability for CTx and osteocalcin were significantly larger than those for TRAP5b and BALP. This marked diurnal variation in five bone markers suggested that the time of blood sampling should be fixed when studying bone marker concentrations in bovine plasma.

Two genotypes among 'Candidatus Mycoplasma haemobos' strains based on the 16S-23S rRNA intergenic spacer sequences.

'Candidatus Mycoplasma haemobos', sometimes causative of bovine infectious anemia at various extents, has been demonstrated throughout the world. Here, we show two distinct types of 'Ca. M. haemobos' are distributed among cattle in Japan, by examining the primary and secondary structures of the 16S-23S rRNA intergenic spacer region that has been shown to be a stable genetic marker for mycoplasma species. Our results may explain differences in severity of anemic condition as well as provide a genetic marker for an epidemiological study of bovine hemoplasma infections.

Estimation of glomerular filtration rate in calves using the contrast medium iodixanol.

To develop a simple procedure for estimating glomerular filtration rate (GFR) in calves, a three-sample method using iodixanol was first compared to that using the standard agent inulin. Iodixanol and inulin were co-administered intravenously to calves at 40 mg I/kg and 40 mg/kg, respectively, and blood was collected 30, 60, 120, and 180 min later. Serum iodixanol and inulin concentrations were separately determined by high performance liquid chromatography and colorimetry. Serum urea nitrogen (UN) and creatinine concentrations were also measured. GFR estimated by iodixanol was consistent with that using inulin in clinically healthy calves. Based on GFR estimations in healthy calves and those renal-loaded with iodixanol, it was found that the serum creatinine concentrations became elevated when GFR decreased to 60% of the reference value. In contrast, serum UN concentrations fluctuated widely, presumably due to extra-renal factors. When GFR was estimated using the three-sample method and compared with the single-blood-sample method, 62/69 (90%) of samples tested were within the agreement plots. The results demonstrated that the single-blood-sample method using iodixanol may be useful in monitoring GFR in calves.

Effect of follicular aspiration at the onset of progesterone-based timed artificial insemination on the follicular dynamics and fertility of early postpartum Japanese black cows.

The effect of gonadotropin-releasing hormone analogue (GnRH-A) or follicular aspiration at the onset of progesterone-based timed artificial insemination (TAI) on subsequent follicular growth and synchronization of ovulation was examined in early postpartum Japanese Black cows. A total of 40 (22 in Exp. 1 and 18 in Exp. 2) Japanese Black cows at 20-30 days postpartum were fitted with a progesterone releasing internal device (PRID) for 7 days, injected with a prostaglandin F2α analogue upon removal of the PRID and GnRH-A 48 h later, and inseminated 18 h after GnRH-A injection. In Exp. 1, the animals were divided into three groups (untreated control, GnRH-A injection or follicular aspiration) of different treatments on the first day of PRID insertion (day 0), and the synchronized ovulation rate in the follicular aspiration group (100%; 8/8) tended to be higher (P = 0.077) than that in the control group (42.9%; 3/7). In Exp. 2, follicular growth in the GnRH (n = 9) and follicular aspiration (n = 9) groups was monitored by ultrasonography. Four out of the nine animals in the GnRH group had a corpus luteum on either day 4 or day 7 (OV group), and the other five animals had no induced ovulation (NOV group). The diameter of the ovulatory follicle on day 9 in the OV group (1.44 ± 0.11 cm) tended to be greater (P = 0.078) than that in the NOV group (1.13 ± 0.07 cm). Follicular aspiration at the onset of PRID-based TAI of early postpartum Japanese Black cows, regardless of the resumption of ovarian cyclicity, tended to result in a higher rate of synchronization of ovulation than that of the untreated controls.

Validation of a direct time-resolved fluoroimmunoassay for progesterone in milk from dairy and beef cows.

The aim of this study was to validate a direct time-resolved fluoroimmunoassay (TR-FIA) for quantifying progesterone concentrations in milk during the bovine oestrous cycle. Holstein-Friesian and suckled and non-suckled Japanese Black cows were used to demonstrate the relationship between milk and plasma progesterone concentrations and to monitor progesterone profiles in milk and plasma during the oestrous cycle. The minimum detection level of the assay was 1.53ng/mL. Progesterone concentrations in milk and plasma changed in a similar manner throughout the oestrous cycle in dairy and beef cows, and milk and plasma progesterone profiles were significantly correlated (P<0.001). The study confirmed that a direct TR-FIA can be used to monitor the oestrous cycle in cattle and to quantify progesterone concentrations in whole milk.

Characteristics of the stimulatory effect of kisspeptin-10 on the secretion of luteinizing hormone, follicle-stimulating hormone and growth hormone in prepubertal male and female cattle.

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and growth hormone (GH) in prepubertal male and female cattle. The experiments were performed from May to June using five male (4-6 months old) and five female (5-6 months old) Japanese Black calves. A single intravenous (iv) injection of Kp10 (5 microg/kg body weight (b.w.): 3.85 nmol/ kg b.w.) significantly stimulated the release of LH and FSH in male and female calves (P<0.05). A single intramuscular injection of Kp10 (5 microg/kg b.w.) also significantly stimulated the release of LH and FSH in male calves (P<0.05), though the response was smaller than that to the iv injection. The injection of Kp10 did not alter the basal plasma concentration of GH in male or female calves. The area under the curve (AUC) of both LH and FSH for a 120-min period after the iv injection of Kp10 was significantly greater in the males than females (P<0.05). These results show that Kp10 can stimulate the release of LH and FSH in calves of both sexes and that the response to the peptide is greater in males at this age. They also show that Kp10 has no effect on the release of GH in male and female calves and that the LH- and FSH-releasing effect of Kp10 is greater after an iv injection than after an im injection in calves.

Effect of suckling on embryo production by repeated ovum pick-up before and after timed artificial insemination in early postpartum Japanese black cows.

We investigated whether suckling would affect embryo production of cows bred by timed artificial insemination (TAI) following an ovulation synchronization protocol combined with ovum pick-up and progesterone releasing intravaginal device (OPU-PRID-TAI protocol). The number of oocytes and transferable embryos collected by repeated OPU, performed before and after TAI, were recorded. A total of 14 Japanese Black cows were divided into weaned (n=7) and suckled groups (n=7). All 14 cows were treated with OPU on day 0 (the first day of treatment) and then with a PRID for 9 days. Prostaglandin F(2alpha) analog was administered on day 7, GnRH analog was administered on day 10 (36 h after removal of the PRID) and TAI was performed 12 h later. Ovulation was confirmed by palpation per rectum the following day. After TAI, additional OPU sessions were performed on days 18, 25 and 32. The synchronized ovulation rates of the weaned and suckled groups were 100 and 85.7%, and the conception rates were 71.4 and 42.9%, respectively. Immature oocytes were fertilized and cultured in vitro. The numbers of oocytes collected and blastocysts generated were similar between the individual OPU sessions in both groups. However, the total numbers of oocytes collected, cultured oocytes, cleavage embryos and blastocysts as well as the proportions of cleavage embryos and blastocysts to cultured oocytes were all significantly (P<0.05) greater in the weaned group compared with the suckled group. These results suggest that the OPU-PRID-TAI protocol has the potential to produce a significant number of good-quality embryos in vitro after repeated OPU in early postpartum weaned Japanese Black cows. To collect more oocytes and produce more embryos, we suggest that calves be removed from cows scheduled for treatment using this protocol.

Applicability of a progesterone-based timed artificilal insemination protocol after follicular fluid aspiration using the ovum pick-up technique in suckled beef cows.

We conducted a progesterone-based timed AI protocol after follicular fluid aspiration using the ovum pick-up (OPU) technique to examine its applicability to the suckled beef cow. A total of 19 beef cows were randomly allocated to one of the following three groups based on the number of days postpartum: 13 to 60 days (Group A: suckled; early postpartum period, n=9), 61 to 150 days (Group B: suckled; mid postpartum period, n=6), or 151 to 281 days (Group C: non-suckled; prolonged open period, n=4) postpartum. These cows were treated with follicular fluid aspiration and insertion of a progesterone-releasing intravaginal device (PRID) on day 0. The PRID was removed and 500 microg of cloprostenol was intramuscularly administered on day 7. A dose (100 microg) of fertirelin acetate was injected intramuscularly 48 hours later, and this was followed by a timed AI (TAI) after another 18 hours (day 10). Serum samples were taken on days 0, 7, 9, 10, 12, 17, 24 and 31 for determination of the estradiol-17beta (E(2)) and progesterone concentrations. Pregnancy diagnosis was made by rectal palpation approximately 60 days after TAI. There was no significant difference in the peripheral E(2) concentrations among the three groups during the period of the hormonal treatment. The average progesterone concentrations in Group A on day 17 were significantly higher than those in Group B and exceeded 1.0 ng/ml on day 17 and thereafter. There was no significant difference in the numbers of collected immature oocytes among the three groups. The pregnancy rates in Groups A, B, and C were 77.8% (7/9), 83.3% (5/6) and 50.0% (2/4), respectively. In conclusion, this timed AI protocol is applicable to suckled beef cows within the period of 60 days postpartum.

Tumor necrosis factor-alpha genetic polymorphism may contribute to progression of bovine leukemia virus-infection.

In a previous report, we had indicated that in a sheep model, the expression of tumor necrosis factor (TNF)-alpha was closely associated with disease progression in sheep experimentally infected with bovine leukemia virus (BLV). However, individual variabilities are observed in these responses in BLV-infected animals. To attempt to identify genetic factors promoting the progression to BLV-induced lymphoma, we endeavored to determine whether there are any polymorphisms in the TNF-alpha gene among 291 individuals and whether this would affect the level of TNF-alpha expression and concomitant progression of BLV-induced disease or increase in the provirus load in the carriers. We found that the frequency of the TNF-alpha -824G allele, which has been associated with low transcription activity of the promoter/predicted enhancer region of the bovine TNF-alpha gene, was higher in individuals with BLV-induced lymphoma than in asymptomatic carrier individuals. In addition, we observed a tendency for increased BLV-provirus load in cattle with TNF-alpha -824G/G homozygote compared to TNF-alpha -824A/A homozygote or TNF-alpha -824A/G. These data suggest that the observed polymorphism in the promoter region of TNF-alpha gene could at least in part contribute to the progression of lymphoma in BLV-infection.

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection.

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-alpha and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-alpha-induced responses, in this study we examined the TNF-alpha-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-alpha (rTNF-alpha) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5+ or sIgM+ cells and these cells showed resistance to TNF-alpha-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-alpha-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.

Comparison of the effectiveness of ovulation synchronization protocol in anestrous and cycling beef cows.

Applicability of ovulation synchronization protocol using GnRH and PGF(2alpha) (PGF) injection to anestrous beef cows remains controversial. We compared the effectiveness of the protocol in the anestrous stage of the beef cow with that in the cycling stage using the same animals. Ovaries of five Japanese Black and three Japanese Shorthorn cows were ultrasonographically examined, and blood samples were collected daily for hormonal analyses. Each animal received the protocol twice (Day -6 to -8: GnRH, Day 0: PGF, Day 2: GnRH). Additional blood samples were taken before and after GnRH injection for LH and FSH measurements to evaluate the pituitary function. For the ovarian status at the onset of the protocol cows were divided into anestrous (n=8) and cycling (n=8) stages. There was no significant difference in size of the dominant follicle at the first and second GnRH injections, and in the magnitude of the pituitary response to GnRH between the two stages. However, the size of the corpus luteum and progesterone concentrations at the PGF injection in the anestrous stage were significantly smaller and lower (P<0.01), respectively, and ovulation synchronization rate in the anestrous stage was significantly lower (P<0.05) than in the cycling stage. In conclusion, ovulation synchronization protocol in anestrous beef cows has limited effectiveness.