Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high‐throughput sequencing

Small RNA represents several unique non‐coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II‐arrested stage (M II) and then sequenced using small RNA high‐throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR‐342 has the largest fold change (9.25‐fold). Six highly expressed miRNAs (let‐7i, miR‐10b, miR‐10c, miR‐143, miR‐146b and miR‐148) were validated by real‐time quantitative PCR (RT‐qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy‐five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division.     

Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket"

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.     

Inflammatory cytokines and antigen presenting cell activation

Immune responses are stimulated in response to threats against health. In animals, defense against infectious agents, particularly rapidly growing viruses and bacteria, requires an immediate response to limit growth and dissemination, and then stimulation of a more prolonged, specific immunity to prevent re-infection. The process by which animals meet the dual needs of an immediate response to danger and initiation of long-term protection is substantially influenced by inflammatory cytokines produced primarily by macrophages and professional antigen presenting cells (APCs). Inflammatory cytokines mobilize the immune system in response to danger and increase the efficiency of an immune response as effectors of APC function. Here we review the evidence for the involvement of inflammatory cytokines in immune induction and as mediators of APC activity, with a particular emphasis on swine and on the induction of immunity at mucosal surfaces. The vast majority of infections occur at mucosal surfaces of the enteric, respiratory and reproductive tracts, and induction of protective immunity at these sites is particularly challenging. Induction of immunity at mucosal surfaces of the small intestine is greatly facilitated by the oral adjuvant, cholera toxin (CT). CT potentiates inflammatory cytokine and costimulatory molecule expression in macrophages, and stimulates humoral and cell-mediated immune responses both locally and systemically. These observations are consistent with the hypothesis that activation of APCs is a key step in the induction of antigen-specific immunity, and that inflammatory cytokine expression is a hallmark of activated APC function. The efficacy of vaccine adjuvants, particularly in the context of mucosal immunity, may be determined by their ability to induce a controlled inflammatory response in gut-associated lymphoid tissue, characterized by the expression of various costimulatory molecules and inflammatory cytokines. Thus, elucidation of the patterns of inflammatory cytokine expression and features of APC activation will help to facilitate the rational development of more efficacious vaccines.     

Force Plate Gait Analysis in Doberman Pinschers with and without Cervical Spondylomyelopathy

Background

The most accepted means of evaluating the response of a patient with cervical spondylomyelopathy (CSM) to treatment is subjective and based on the owner and clinician's perception of the gait.

Objective

To establish and compare kinetic parameters based on force plate gait analysis between normal and CSM‐affected Dobermans.

Animals

Nineteen Doberman Pinschers: 10 clinically normal and 9 with CSM.

Methods

Force plate analysis was prospectively performed in all dogs. At least 4 runs of ipsilateral limbs were collected from each dog. Eight force platform parameters were evaluated, including peak vertical force (PVF) and peak vertical impulse (PVI), peak mediolateral force (PMLF) and peak mediolateral impulse, peak braking force and peak braking impulse, and peak propulsive force (PPF) and peak propulsive impulse. In addition, the coefficient of variation (CV) for each limb was calculated for each parameter. Data analysis was performed by a repeated measures approach.

Results

PMLF (P = .0062), PVI (P = .0225), and PPF (P = .0408) were found to be lower in CSM‐affected dogs compared with normal dogs. Analysis by CV as the outcome indicated more variability in PVF in CSM‐affected dogs (P = 0.0045). The largest difference in the CV of PVF was seen in the thoracic limbs of affected dogs when compared with the thoracic limbs of normal dogs (P = 0.0019).

Conclusions and Clinical Importance

The CV of PVF in all 4 limbs, especially the thoracic limbs, distinguished clinically normal Dobermans from those with CSM. Other kinetic parameters less reliably distinguished CSM‐affected from clinically normal Dobermans.     

Application of an ELISA Milk Pregnancy Test in Beef Cows

Pregnancy‐associated glycoproteins (PAG) are secreted by the binucleate giant cells of the ruminant placenta and enter maternal circulation at the time of placental attachment. The IDEXX Milk Pregnancy Test (IDEXX, Westbrook, ME) detects a subset of PAG in milk. Although designed as a management tool for dairy cows, there is potential for using the milk PAG test in beef cows. Our objective was to compare the performance of the milk PAG ELISA with a gold standard method for pregnancy diagnosis and determine the agreement between milk and serum PAG analysis in lactating beef cows. Angus and Angus‐crossed cows (n = 332) from two Michigan beef herds were enrolled in this study. Cows were subjected either to timed artificial insemination followed by exposure to a bull or exclusively exposed to a bull. The bulls and cows were separated 30 days prior to examination. Serum and milk samples were collected and submitted within 24 h of collection to a commercial laboratory for PAG analysis using the IDEXX Milk Pregnancy Assay (milk) and the IDEXX Bovine Pregnancy Assay (serum). Concurrently with milk and serum collection, each cow was examined transrectally by palpation or ultrasonography. When compared to transrectal examination, the performance (and 95% confidence intervals) of the milk PAG ELISA was sensitivity of 99.7% (99.0–100.0%) and specificity of 80.8% (65.6–95.9%). The lower specificity is likely due to the low prevalence (9.9%) of open cows (n = 30) in the herds examined. Of the 332 cows examined, 1.8% (n = 6) were classified as rechecks using the milk PAG ELISA. Results of the milk and serum PAG ELISA were in high agreement (kappa coefficient = 0.91). The milk PAG ELISA was accurate in predicting pregnancy status using milk collected from beef cattle between days 37 and 125 post‐insemination and may be useful for aiding management decisions in beef herds.