Development and characterization of an immortalized nasopharyngeal epithelial cell line to explore airway physiology and pathology in yak (Bos grunniens).

Airway epithelial cells play a crucial role in investigating the physiological and pathological mechanisms of the respiratory tract in yaks, a species whose unique respiratory system has garnered extensive interest. Despite this growing interest, there currently are no available airway epithelial cell lines from yaks, underscoring the crucial need to establish a yak respiratory epithelial cell line. Therefore, our objective was to isolate a population of primary yak nasopharyngeal epithelial cells (pYNE) and transform them into immortalized yak nasopharyngeal epithelial cells (iYNE), assessing their suitability as an in vitro model. Employing a combined method of physical elimination and differential adhesion, we successfully isolated a population of high-purity pYNE, and developed an iYNE line through pCI-neo-hTERT plasmid transfection. Karyotype and transmission electron microscopy analyses confirmed that pYNE and iYNE share identical morphologies and structures. Gel electrophoresis and real-time PCR analyses demonstrated that pYNE and iYNE expressed similar levels of KRT18 and CDH1 genes (p ≥ 0.541). Notably, iYNE expressed a significantly high level of TERT gene expression (p < 0.001). Immunofluorescence analysis demonstrated that both cell types expressed Pan-Cytokeratin, ZO-1, and E-cadherin proteins. Furthermore, immunoblotting analysis indicated significantly higher levels of hTERT and Ki67 proteins in iYNE (p < 0.001), and similar levels of Cluadin-3 and Occludin proteins (p ≥ 0.103). Proliferation curve analysis highlighted iYNE's serum-dependency and significantly enhanced proliferation capacities (p < 0.001). Additionally, pYNE and iYNE cells demonstrated comparable susceptibilities to infectious bovine rhinotracheitis virus (IBRV). These findings collectively suggest that the developed iYNE retains the evaluated physiological characteristics of pYNE, making it an appropriate in vitro model. This advancement will facilitate further investigation into the respiratory physiological and pathological mechanisms in yaks.

Multi-omics reveals that alkaline mineral water improves the respiratory health and growth performance of transported calves.

BACKGROUND: Long-distance transportation, a frequent practice in the cattle industry, stresses calves and results in morbidity, mortality, and growth suppression, leading to welfare concerns and economic losses. Alkaline mineral water (AMW) is an electrolyte additive containing multiple mineral elements and shows stress-mitigating effects on humans and bovines. RESULTS: Here, we monitored the respiratory health status and growth performance of 60 Simmental calves subjected to 30 hours of road transportation using a clinical scoring system. Within the three days of commingling before the transportation and 30 days after the transportation, calves in the AMW group (n = 30) were supplied with AMW, while calves in the Control group (n = 29) were not. On three specific days, namely the day before transportation (day -3), the 30th day (day 30), and the 60th day (day 60) after transportation, sets of venous blood, serum, and nasopharyngeal swab samples were collected from 20 calves (10 from each group) for routine blood testing, whole blood transcriptomic sequencing, serology detection, serum untargeted metabolic sequencing, and 16S rRNA gene sequencing. The field data showed that calves in the AMW group displayed lower rectal temperatures (38.967 ℃ vs. 39.022 ℃; p = 0.004), respiratory scores (0.079 vs. 0.144; p < 0.001), appetite scores (0.024 vs. 0.055; p < 0.001), ocular and ear scores (0.185 vs. 0.338; p < 0.001), nasal discharge scores (0.143 vs. 0.241; p < 0.001), and higher body weight gains (30.870 kg vs. 7.552 kg; p < 0.001). The outcomes of laboratory and high throughput sequencing data revealed that the calves in the AMW group demonstrated higher cellular and humoral immunities, antioxidant capacities, lower inflammatory levels, and intestinal absorption and lipogenesis on days -3 and 60. The nasopharynx 16S rRNA gene microbiome analysis revealed the different composition and structure of the nasopharyngeal microflora in the two groups of calves on day 30. Joint analysis of multi-omics revealed that on days -3 and 30, bile secretion was a shared pathway enriched by differentially expressed genes and metabolites, and there were strong correlations between the differentially expressed metabolites and the main genera in the nasopharynx. CONCLUSIONS: These results suggest that AMW supplementation enhances peripheral immunity, nutrition absorption, and metabolic processes, subsequently affecting the nasopharyngeal microbiota and improving the respiratory health and growth performance of transported calves. This investigation provided a practical approach to mitigate transportation stress and explored its underlying mechanisms, which are beneficial for the development of the livestock industry. Video Abstract.

Effects of meloxicam on the welfare of Holstein calves from 6 weeks to 6 months old undergoing amputation dehorning.

Amputation dehorning (AD) is a common practice performed on calves, causing harmful effects such as pain, distress, anxiety, and fear. These effects extend to behavioral, physiological, and hematological responses, prompting serious ethical concerns regarding animal welfare, even when performed with local anesthesia. Meloxicam, a nonsteroidal anti-inflammatory drug, has been widely used to mitigate the side effects of dehorning and disbudding in calves. However, there is a notable gap in research regarding the effects of meloxicam on calves aged 6 wk to 6 mo undergoing AD procedures. This study was designed to assess the effectiveness of co-administering meloxicam with lidocaine, a cornual nerve anesthetic, in alleviating the adverse effects caused by the AD procedure in calves within this age range, compared with the use of lidocaine alone. Thirty Holstein calves were enrolled and randomly divided into 2 groups. The first group received a subcutaneous injection of 5 mL of lidocaine in the horn area and a subcutaneous injection of 0.9% saline at a dose of 0.025 mL/kg in the neck, administered 10 min before the AD procedure. The second group received a combination of lidocaine and meloxicam: a subcutaneous injection of 5 mL of lidocaine in the horn area and a subcutaneous injection of 20 mg/mL meloxicam at a dose of 0.025 mL/kg in the neck, also administered 10 min before the AD procedure. To avoid subjective bias, the researchers were blinded to the treatment groups. Pain-related behaviors, including tail flicking, head shaking, ear flicking, head rubbing, head crossing bar, and kicking, were observed, and physiological parameters, including heart rate, rectal temperature, respiration rate, mechanical nociceptive threshold (MNT), daily active steps, and food intake were monitored. Hematological conditions were determined using enzyme-linked immunosorbent assays and routine blood tests. The data were processed using a generalized linear mixed model. The outcomes demonstrated that the AD procedure increased the frequencies of ear flicking and resulted in rises in the respiration rate, heart rate, rectal temperature, and daily active steps. It also led to decreases in total food intake, forage intake, hay intake, MNT, and increased concentrations of prostaglandin E2 (PgE2), IL-1ß, tumor necrosis factor-α (TNF-α), nitric oxide (NO), and malondialdehyde, as well as glutathione peroxidase activity. However, calves that received meloxicam treatment showed significant improvements in response to the AD procedure, including lower respiration rates, heart rates, and rectal temperatures; higher MNT; and lower intermediate cell ratio. They also had higher red blood counts, hemoglobin levels, hematocrit values; larger mean platelet volumes; and lower concentrations of PgE2, IL-1ß, TNF-α, and NO. These results suggest that co-administration of lidocaine and meloxicam may aid in mitigating the adverse effects induced by the AD procedure on these calves, thereby supporting the use of meloxicam in conjunction with a local anesthetic in AD procedures for calves aged 6 wk to 6 mo.