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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.

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Abstract. This study was carried out to evaluate the blood profile and tissue expression of Anti-Müllerian hormone (AMH) as a biomarker for granulosa-theca cell tumors (GTCTs) in cattle. Five cases with unilateral ovarian GTCTs (GTCT group) were investigated in comparison to other groups of Japanese Black cows, which had either cystic ovarian disease (COD group, n=5), a functional corpus luteum on Days 9 to 11 of the estrous cycle (Day 0=estrus; CL group, n=13) or received superovulation treatment (SOT group, n=13). We used transrectal ultrasonography and measured plasma AMH, estradiol-17β (E 2 ), progesterone (P 4 ) and testosterone (T) levels. Moreover, GTCT tissues were collected and examined by immunohistochemical staining (IHC) for AMH. In the GTCT group, ultrasound images of GTCTs were variable and not definitive. However, the AMH level in the GTCT group (n=3, 58.1 ± 66.3 ng/ml) was significantly higher than in the COD, CL and SOT groups (0.1 ± 0.1 ng/ml for GTCT vs. COD, P<0.05; 0.2 ± 0.1 and 0.3 ± 0.2 ng/ml, respectively for GTCT vs. CL and SOT, P<0.01). The other hormonal levels in the GTCT group had no significant differences compared with the COD or SOT group. Neoplastic granulosa cells labeled with AMH antibody clearly demonstrated a variety of tissue patterns in all cases by IHC. To the best of our knowledge, this is the first study to investigate the blood profile and IHC of AMH in bovine GTCTs. Our findings indicate that AMH may be a novel biomarker to diagnose GTCTs in cattle. Key words: Anti-Müllerian hormone (AMH), Cattle, Granulosa-theca cell tumors (GTCTs), Immunohistochemical staining (J. Reprod. Dev. 58: [98][99][100][101][102][103][104] 2012) G ranulosa-theca cell tumors (GTCTs) are the most common ovarian tumors in cattle [1], and the incidence may be less than 0.5% [2]. GTCTs can affect various breeds [3] and occur at various ages [4] in cattle. GTCTs produce a variety of steroid hormones, causing a subsequent elevation of plasma estradiol-17β (E 2 ), progesterone (P 4 ) and/or testosterone (T) levels [4]. Therefore, a variety of clinical signs, such as nymphomania [5], virilism [6] and mammary gland development [7], could be presented. In contrast, some in GTCTs may not result in abnormal reproductive behavior at all [3]. Preliminary diagnosis of GTCTs can be achieved by transrectal palpation and ultrasonography [1,4]. GTCTs should be suspected if a chronic cystic ovarian disease (COD) does not respond to standard treatment regimens or if the diameter of the ovary is more than 100 mm [1]. Thus, GTCTs should be distinguished from other conditions, such as COD, oophoritis, ovarian abscesses and parovarian cysts, by these clinical signs [8]. However, the distinctions might be difficult depending on the clinical signs and other diagnostic methods, so a definitive diagnosis can only be made based on histopathological examination of the affected ovary [3]. In the treatment of unilateral bovine GTCTs, unilateral ovariectomy is only indicated in cows that do not exhibit alterations in secon...

Endometritis is one of the major diseases causing infertility in the cow. Intrauterine infusion of povidone-iodine (PVP-I) is a common treatment. However, the optimal concentration of PVP-I for treating endometritis effectively remains unknown. We tested concentrations of 2.0% or 0.5% PVP-I for treating clinical endometritis in dairy cattle. In Experiment 1, bacteria isolated from the uterus were incubated with either 2.0% or 0.5% PVP-I, and the numbers of bacterial colonies were counted. In Experiment 2, 18 cows with clinical endometritis were treated with either 2.0% or 0.5% PVP-I (n=9 in each group). Cytology samples and bacteria were collected using a cytobrush on weeks 0 (W0), 1 (W1) and 2 (W2) after treatment. Subsequent reproductive performance was compared between the two groups. In Experiment 1, both concentrations had a similar antiseptic outcome. In Experiment 2, the percentage of polymorphonuclear neutrophils (PMN%) in the endometrial epithelium at W2 in the 2.0% group was significantly lower (P<0.05) than in the 0.5% group, although the PMN% decreased significantly from W0 to W2 (P<0.01) in both groups. Decreases in bacterial infection rates from W0 to W2 were similar in both groups. The first service conception rate was higher, numbers of services per conception were fewer, and time to conception was shorter in the 2.0% group than in the 0.5% group. Thus, an intrauterine infusion of 2.0% PVP-I was better than 0.5% in treating clinical endometritis in these dairy cattle.

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The current study aimed to define the plasma profile of anti-Müllerian hormone (AMH) and follicle stimulating hormone (FSH) in heifers during postnatal life until achieving puberty, as defined by plasma progesterone (P4) profile, to demonstrate a relationship between AMH and age of puberty onset. Blood samples collected from 11 Japanese Black female calves within 1 week after birth (W 0) and then biweekly until the sixth week after puberty (WP 6) were assayed for AMH, FSH, and P4. The heifers were classified into two groups based on age of puberty onset: ≤42 weeks (early puberty group; EP, n = 4) and ≥44 weeks (late puberty group; LP, n = 7). Minimal plasma AMH concentration occurring at W 0 gradually increased to its peak level by W 10 (fourfold higher than W 0; P < 0.01) before gradually declining to a steady plateau 6 weeks before puberty (WP -6). The AMH peak was preceded by a significant rise in plasma FSH at W 4, W 6, and W 8 compared with W 0. Plasma AMH at W 16 positively correlated with WP 4 and WP 6 (r = 0.69 and 0.71, respectively; P < 0.05). Overall plasma AMH and FSH was significantly higher and lower in EP compared with LP, respectively. In conclusion, heifers exhibit a characteristic plasma AMH profile during postnatal life, such that plasma AMH at an early prepubertal age could be a biomarker for precocious puberty and postpubertal AMH levels.

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 miR-NAs 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. DatabasesThe 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).