Incidence of Subclinical Ketosis and Evaluation of Associated Risk Factors in Early Lactating Dairy Buffaloes

Muhammad Zaeem Abbas1, Khurram Ashfaq1, Amjad Islam Aqib2, Mughees Aizaz Alvi1*, Muhammad Haleem Tayyab1, Muzafar Ghafoor1, Muhammad Usman Naseer3, Ali Abbas4 and Ali Hassan1 1Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad 2Department of Medicine, Cholistan University of Veterinary and Animal Sciences, Bahawalpur 3Department of Parasitology, University of Agriculture, Faisalabad 4Veterinary Research Institute, Lahore Cantt, Lahore Article Information Received 04 June 2020 Revised 11 July 2020 Accepted 10 November 2020 Available online 13 December 2021 (early access)

O wing to high population of cattle (~51.5 million) and buffalo (~42.4 million), Pakistan stands among the highest milk producers (Pakistan Economic Survey, 2020). Pakistan livestock section attributes approximately 57,890 thousand tons milk per annum with an average of 1.46 L/animal/day. Compared to the number of livestock animals and agricultural land, theses production marks are considered low and substandard according to international standards. This suboptimum milk production is not only due to the poor genetic potential but also caused by a variety of infectious and metabolic diseases. Among various metabolic diseases, ketosis is a common metabolic disease that is characterized by the imbalance metabolism of carbohydrate and volatile fatty acids (VFAs). During early lactation periods, animals produce more milk than glucose/glycogen are fed, and this led to negative energy balance that causes mobilization of body fat reserves in order to meet the body requirements. The mobilization of fat leads to production of fatty acids at high rates which are responsible for ketone bodies formation. The high level of ketone bodies formation leads to development of ketosis (Radostits et al., 2007).
The salient features of this disease are ketonemia, ketonuria, hypoglycemia and low level of hepatic glycogen (Tehrani-Sharif et al., 2012). Clinical ketosis typically appears within the first six to eight weeks after calving and results in anorexia, licking, blindness, hard dry faeces, rapid loss of condition and decreased milk production (Youssef et al., 2010). Subclinical ketosis (SCK) are accompanied by decreased milk yield and lower milk protein and lactose contents (Vanholder et al., 2015). Ketosis prone animals to delayed oestrus, low conception rate, increased calving intervals, cystic ovarian disease and mastitis (Radostits et al., 2007).
There are number of risk factors that can lead to ketosis such as dystocia, retention of foetal membrane, high milk production capacities, puerperal metritis and milk fever (Vanholder et al., 2015). While the condition is common, limited information is available in dairy buffaloes, which comprise about 60% of the total lactating O n l i n e

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herds of Punjab province of Pakistan. Therefore, the present study was conducted to assess the occurrence and prevalence of ketosis in buffaloes. Additionally, we aimed to evaluate associated risk factors which can alter the ketosis in lactating buffaloes.

Materials and methods
This study encompassed different dairy farms in the vicinity of district Faisalabad. Blood, milk and urine samples were collected from a total of 96 buffaloes from dairy farms. The study was approved by Faculty Board and Graduate Studies for all kinds of ethical norms of research notification vide letter No. CE 841/15/08/15. Milk and urine samples from all quarters of buffaloes were collected in separate sterile screw-caped bottles. Both samples were analysed for ketosis by Ross Modification of Rothera's Test (Farooq, 2014). Blood was taken from jugular vein and analyzed by ketometer (Abbott Precision Xtra™) to detect the level of ketone bodies (Iwersen et al., 2009) according to the manufacturer's instructions. A cut-off point (≥1.2mmol/L) was used to differentiate between positive and negative samples (Iwersen et al., 2009). Prevalence was estimated at 95% confidence interval (CI). Chi square test was used to analyze the data collected (Schork and Remington, 2010) to perform test of association between variables and results were significant at p<0.05.

Results and discussion
Analysis of the ketosis, determined by the ketometer, demonstrated a positivity rate of 8.33% (8/96) in total collected and analyzed samples. Further analysis of the disease occurrence across the lactation period indicated a high incidence rate during the third (13.3%) and fourth week (11.1%) post-partum. The buffaloes carrying body condition score 4 (15%) and >4 (20%) were found to have higher ketosis. The body condition scoring was performed as described previously (Radostits et al., 2007). The ketosis was found to be higher in 8-9 (11.1%) and 10-11 (10.0%) years of age. Likewise, animals with 18 and 16 liters of milk yield were found more ketoic with 33.3% and 10.0%, respectively. Regarding parity number, buffaloes during 3 rd and 4 th parity carried 11.1% and 10.5% ketosis, respectively. The association of postpartum duration was found to be statistically non-significant (χ 2 =1.520, p-value= 0.971). There was a significant (p<0.05) co-relation with ketosis in milk yield and body condition score while nonsignificant (p>0.05) relationship was found between age, postpartum duration, and parity (Table I). Significant (p<0.05) relation was found with puerperal metritis while non-significant (p>0.05) relationship was identified between mastitis, dystocia and retained placenta (Table II).
The current findings corelated with various studies where ketosis prevalence was ranged from 0% to 33.9% in the first two months of lactation (Dohoo and Martin, 1984;Andersson and Emanuelson, 1985;Nielen et al., 1994;Duffield et al., 1997). First eight weeks of postpartum was considered suitable to check the prevalence of ketosis. It was recorded high during the third (13.3%) and fourth week (11.1%) of lactation. Findings of this study are in line with the previous studies showing that the peak prevalence of subclinical ketosis occurs in the first 2-4 weeks of lactation (Duffield et al., 1997(Duffield et al., , 1998 which is probably due to high milk production in these weeks. The association of body condition score (BCS) was statistically significant (χ 2 = 8.104, p-value = 0.044). Buffaloes having BCS 3.5-4 and >4 were more ketoic with a prevalence of 15% and 20%, respectively. It has previously been reported that BCS at calving higher than 3.25 or 3.5 on a 5-point scale was associated with a higher risk of development of ketosis (Gillund et al., 2001;Busato et al., 2002). This may be due to animals having high BCS at pre-calving carry lower dry matter intake (DMI). Such animals eat less at calving than thin cows which may lead to high concentrations of non-esterified fatty acids (NEFA) in plasma and high negative energy balance postpartum leading to the development of ketosis (Vanholder et al., 2015). The high milk production causes intensive negative energy balance, increased hepatic ketogenesis and fat mobilization which cumulatively result in ketosis (Duffield, 2000;Detilleux et al., 1994). The buffaloes in 3 rd (11.1%) and 4 th (10.5%) parity were showed higher ketosis. The association of parity was found statistically non-significant (χ 2 =1.317, p-value=0.971). The study findings are in line with already published reports. The chances of ketosis increase with the increase in parity. This is attributed to high milk production in this period that leads to high prevalence of ketosis (Vanholder et al., 2015). In this study, the peak prevalence of ketosis was recorded in 8-9 (11.1%) and 10-13 (10%) years of age. It was observed that association of age was statistically non-significant (χ 2 =0.938, p-value=0.800). In a couple of studies, it has been documented that the likelihood of ketosis increases with the increase in age (Vanholder et al., 2015). Among eight ketosis positive buffaloes, three were mastitis positive. In our current study, the association of mastitis was found statistically non-significant (χ 2 =0.108, p-value=0.742). The buffaloes with mastitis have 1.28 times more chances of ketosis as compared to mastitis negative animals. Previously, it has been reported that mastitis increases the chances of ketosis in high producing animals. This discrepancy could be attributed to the impairment of udder defence mechanisms during hyperketonemia (Suriyasathaporn et al., 2000).

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It was observed that animals with dystocia have 1.50 times higher risk of ketosis in the recent lactation as has been reported by Anna and Vertenten (2014). Notably, no significant increased ketosis was noticed in dairy animals having dystocia and twins, however, only two buffaloes carried retained placenta among eight ketosis positives animals. Collectively, in this study, no non-significant association between ketosis and retained placenta was noticed as previously described by Vanholder et al. (2015).
However, there was 1.296 times more chances of ketosis in buffaloes having retention of placenta, whereas metritis positive animals carried 4.680 times higher chances of ketosis compared to metritis negative animals. It has been reported in previous studies that milking animals in early lactation with subclinical ketosis had an increased risk of metritis (Dohoo and Martin, 1984). However, most studies including ours, proposed that ketosis is an outcome rather than a cause of the metritis.

Conclusion
Taken together, the presents study found a higher prevalence of ketosis in buffalos. It was noticed that animals having high milk yield, being in the third week of lactation, having 3.5-4 body condition score, and in the third parity are more prone to ketosis compared to normal and healthy animals.