| In recent years,with the intensified development of dairy farming,there has been a continuous improvement in the individual milk production level of cows.During the peripartum period of dairy cows,due to the inadequate adjustment of dietary nutrition or excessive milk production,the body experiences an energy imbalance,leading to the occurrence of metabolic disorders such as ketosis.Ketosis,as a common metabolic disease during the peripartum period,severely affects the production and reproductive performance of dairy cows.The circadian clock is an inherent regulatory system in mammals that is involved in regulating various physiological processes,including energy metabolism,immune defense,oxidative stress,and reproductive endocrine functions,through circadian rhythm changes.The occurrence of ketosis in dairy cows is closely related to pathological processes such as energy metabolism imbalance,oxidative stress,and immune dysfunction.However,it is currently unclear whether the circadian clock system of dairy cows undergoes changes before and after the onset of ketosis,and whether this system is involved in regulating the immune and oxidative stress functions of dairy cows is still unclear.Therefore,investigating the diurnal variations in energy metabolism,immunity,and other related physiological functions in both healthy and ketotic dairy cows is of crucial importance in comprehensively understanding the pathogenesis of ketosis in cows from a circadian rhythm perspective.This understanding will ultimately contribute to more precise and efficient prevention and control strategies for ketosis in cows.Located at latitude 35-39 degrees north,Ningxia enjoys a favorable climate and is widely recognized as a "golden milk source region" within the industry.In recent years,with strong government support,the scale of dairy farming in this region has rapidly expanded.This study collected information on 1,618 lactating cows from a large-scale breeding farm in Ningxia throughout the year 2021.The annual incidence rate of ketosis on the farm was recorded,and the relationship between ketosis incidence and factors such as season,parity,body condition score,body temperature,urine p H,305-day milk yield,lactation curve,and various postpartum diseases was analyzed.Simultaneously,blood samples were collected from a group of cows with the same parity,including five healthy cows and five cows with ketosis,at four time points within a 24-hour period(09:00,17:00,01:00,and 09:00).Using methods such as blood glucose/beta-hydroxybutyrate meters,ELISA kits,and real-time quantitative PCR,metabolic indicators related to glucose and lipid metabolism,oxidative stress levels,inflammatory factor contents,as well as the diurnal variation and differences in the expression of clock genes in the blood of healthy and ketotic cows were measured.The research findings are as follows:1.The investigation results of ketosis show that the annual incidence rate of subclinical ketosis in the dairy cows on the farm in 2021 was 32.90%,while the clinical ketosis incidence rate was 9.53%.The impact of different seasons on the incidence rates of subclinical and clinical ketosis followed a consistent trend,with higher rates observed in the spring and summer seasons compared to the autumn and winter seasons.The influence of parity on the incidence rates of subclinical and clinical ketosis was similar,with higher rates observed in cows with higher parity.Among the cows that experienced subclinical and clinical ketosis,42.86% and 87.50% of the cows,respectively,had a body condition score greater than 3.9.Additionally,67.70% and 59.0% of the cows,respectively,had a body temperature below38.5°C,indicating that cows with a body condition score greater than 3.9 were more susceptible to ketosis,and cows with ketosis had lower body temperatures.The 305-day milk yield of cows with clinical ketosis was significantly lower than that of healthy cows(P < 0.05).By plotting the lactation curve,it was observed that the peak milk production day of cows with subclinical ketosis occurred 0.9 days earlier than that of the healthy group,with a peak milk yield 3.74 kg higher.In contrast,the peak milk production day of cows with clinical ketosis occurred 2.99 days later than that of the healthy group,with a peak milk yield 6.5 kg lower.These findings suggest that high-yielding cows are more prone to subclinical ketosis and that clinical ketosis leads to a significant decrease in milk production.The chi-square test results indicate a significant positive correlation between the incidence of clinical ketosis and the occurrence of digestive diseases and mastitis.Therefore,to prevent the occurrence of ketosis,the influence of factors such as season,parity,and body condition score should be comprehensively considered in the dairy cattle management process.Based on the actual situation,a scientifically adjusted feeding and management plan should be implemented.This is of significant importance for enhancing dairy cow lactation performance,improving milk quality,and effectively ensuring the health and well-being of dairy cattle.2.The results of the ELISA kit analysis revealed that the blood β-hydroxybutyrate(BHBA)levels in the ketotic group were significantly higher than those in the healthy group.However,there was no significant diurnal variation in BHBA levels between the ketotic and healthy cows.The blood glucose levels in the ketotic group were significantly lower than those in the healthy group(P < 0.05),and there was a significant diurnal variation in blood glucose levels between the healthy and ketotic cows(P = 0.0013).The cholesterol levels in the ketotic group were significantly lower than those in the healthy group(P < 0.0001).While the blood cholesterol levels in the healthy cows exhibited a significant diurnal variation,the ketotic cows lost the diurnal variation in blood cholesterol levels.These findings suggest an imbalance in glucose and lipid metabolism in ketotic cows.Although the blood glucose levels in ketotic cows still exhibited significant diurnal variation,the diurnal variation in cholesterol metabolism was lost.3.The results of the oxidative stress analysis showed that both the healthy and ketotic cows exhibited significant diurnal variations in plasma hydrogen peroxide levels,malondialdehyde(MDA)levels,catalase(CAT)activity,and superoxide dismutase(SOD)activity(P = 0.0073,P < 0.0001,P = 0.0079,P = 0.0048).The ketotic group had significantly higher MDA levels,glutathione peroxidase activity,and β-N-acetylglucosaminidase levels compared to the healthy group(P < 0.01,P < 0.0001,P < 0.001,P < 0.0001).The CAT activity in the plasma of ketotic cows was significantly lower than that in the healthy group(P < 0.05).These results suggest that during ketosis in cows,there is an imbalance in the body’s oxidative and antioxidant systems,resulting in elevated oxidative stress levels.However,diurnal variations in oxidative stress levels still exist in the blood of ketotic cows.4.The results of real-time fluorescence quantitative PCR showed that the m RNA expression levels of circadian clock genes BMAL1,NR1D1,RORA,and PER2 in the blood leukocytes of healthy cows also showed significant diurnal variations(P = 0.0082,P < 0.0080,P = 0.0016,P = 0.0030).After the occurrence of ketosis in cows,the expression of BMAL1 and RORA genes in the white blood cells changed.The m RNA expression levels of inflammatory factors IL-10,IL-1β,IL-6,IL-8,and CCL5 in white blood cells of healthy cows exhibited significant diurnal variations(P = 0.018,P < 0.0001,P = 0.0050,P = 0.0027,P =0.0009).Compared to the healthy group,the expression pattern of inflammatory factor genes(IL-10,IL-1β,IL-6,IL-8,and TNF-α)in the blood leukocytes of ketotic cows was altered.These results indicate that the expression levels of inflammatory factors and circadian clock genes in peripheral blood leukocytes of healthy cows exhibit significant diurnal variations,and the diurnal expression patterns of inflammatory factors and circadian clock genes are significantly altered after the occurrence of ketosis,suggesting a potential link between the circadian clock and the development of ketosis.The decreased immune function caused by ketosis may be associated with abnormal circadian clocks in immune cells.Based on the survey data,this study revealed that season and parity significantly influence the incidence of ketosis in dairy cows.Factors such as body condition score and body temperature show a significant correlation with the occurrence of ketosis in cows.The occurrence of clinical ketosis reduces milk production and increases the risk of digestive disorders and mastitis.These findings provide a preliminary basis for further improving the feeding management measures in dairy farms.Furthermore,this study discovered that the metabolic function of glucose and lipids in cows,as well as the oxidative and antioxidative systems,may be regulated by the circadian rhythm.Ketosis disrupts the balance of the oxidative and antioxidative systems in cows,and the decreased resistance caused by ketosis may be associated with the disrupted circadian expression of immune-related factors in peripheral blood leukocytes.These research findings provide a theoretical basis for the accurate diagnosis and precise control of ketosis in dairy cows from a chronobiological perspective. |
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