Physiological, Hematological, Biochemical and Thermographic Changes on Supplementation of Rumen Protected Methionine and Choline in Transition Surti Buffaloes

Transition period is associated with highest incidence of metabolic and infectious diseases in dairy animals (Batisel et al., 2017b). Dairy animals undergo a sequence of stressful events during this period (Sun et al., 2016). Increased nutritional requirement in last trimester owing to fetal growth and during early lactation is further aggravated by low feed intake thus inducing stress (Zhou et al., 2016) and creating negative energy balance (NEB). NEB decreases BCS by mobilizing fat. During transition reproductive organs and liver are highly susceptible to injury predisposing to infection and inflammatory responses. Inflammatory process switches the acute phase response to synthesis of positive acute phase protein to alter the liver function to re-establish homeostasis (Batisel et al., 2017b). Increased body temperature and anorexia may ensue as inflammatory response.

Methionine and choline have metabolic roles for maintaining health during transition period. They enhance hepatic fat metabolism and minimize fatty liver and ketosis. They act as methyl donors and helps in synthesis of each other. Methionine minimizes inflammatory processes by triggering acute phase protein synthesis. Bioavailability of these from diet is limited due to microbial degradation in rumen (Zhou et al., 2016) thus they are coated with resistant material. Methionine levels in common feedstuffs are usually lower than that found in milk of dairy cows. Therefore, routine diet fails to supply metabolizable methionine in optimum amount to dairy animals. Present study was therefore planned to supplement rumen protected methionine and choline (RPM and RPC) in diet during transition period in Surti buffaloes.

Present study was approved by IAEC (066-VCN-VPY-2018) and conducted at Dept. of Veterinary Physiology and Biochemistry, Veterinary College, NAU, Navsari (Gujarat). Based on BCS, previous lactation yield and parity; twenty-seven multiparous Surti buffaloes in advanced gestation were divided in 3 groups as G-I, G-II and G-III with 9 buffaloes in each. G-I (control) was offered basal diet. Basal diet of G-II was supplemented with rumen protected methionine (RPM)@10 gm/buffalo/day and of G-III with both rumen protected methionine@10 gm/buffalo/day and rumen protected choline (RPC) @50 gm/buffalo/day. Dietary supplementation was done for 45 days (-15 days to +30 days of parturition). Feed samples were collected for proximate analysis.

Collection of blood samples (with and without anticoagulant K3EDTA), recordings of meteorological variables (temperature-humidity index by NRC, 1971), body condition scoring (BCS) (Edmonson et al., 1989), physiological parameters (rectal temperature by clinical thermometer-Rossmax Swiss GnbH), tympanic temperature (tympanic thermometer-BPLAccuDiGiT), respiration rate-observing left flank movements) and infrared thermography were done at -15 day, 1^st, 3^rd and 6^th week of calving. Hematological analysis was done using fully automated hematology cell counter machine (MEDONIC-CA-620/530VET). Plasma and serum were separated and stored for further analysis. Randox kits for blood biochemical metabolites, bovine specific ELISA assay kits (Labor-Diagnostica-Nord-GmbH and Co,KG,Nordhorn) for hormones, flame photometer 128 (SYSTRONICS) for electrolytes and atomic absorption spectrophotometer (Model:AAS141,ECIL) for trace elements were used their analysis. Infrared thermography was done using camera FLIR-T420 (FLIR Systems Commercial co., USA).

Data was analyzed statistically by ANOVA using DMRT. Means were compared at P<0.05 (Snedecor and Cochran, 1994).

Meteorological conditions
 
Minimum and maximum ambient temperature (°C) was 22.8 and 34.8, relative humidity was (%) 36.00 and 85.00 and THI was 65.06 and 87.77 respectively.
 
Chemical composition of feed
 
Composition (Table 1) was in normal range (ICAR, 2013).


 
Physiological, BCS and hematological parameters
 
Results for physiological parameters and BCS are presented in Table 2 and for hematological parameters in Table 3.





RT and TT were significantly (P<0.05) lower at 1^st week postpartum in GP-III and GP-II than GP-I whereas no effect was observed for RR.BCS was significantly (P<0.05) higher in both treatment groups at 1^st and 3^rd week of calving than control. Highest BCS was observed at 3rd week postpartum in GP-III.

Supplementation of RPM and RPC reduces calving stress and minimizes inflammatory processes during transition period (Batisel et al., 2017b). This may explain body temperature lowering at 1^st week postpartum in GP-III and GP-II. Calving and early lactation stress aggravates negative energy balance with excessive fat mobilization and loss of BCS as in GP-I of present study. Higher BCS in treatment groups may be due to higher DMI due to RPM and RPC supplementation.
 
Serum biochemical parameters
 
Results for biochemical metabolites, enzymes, hormones, macro and micro-elements are presented in Table 4, 5, 6 and 7 respectively.








 
Metabolites
 
Results show highest (P<0.05) postpartum glucose concentration in GP-III followed by II and I. Similarly, postpartum TP level was significantly (P<0.05) higher in GP-III and GP-II than control. GP-III had highest (P<0.05) TP level during 1^st  week postpartum than GP-II. ALB concentration was significantly (P<0.05) high in both supplemented group during 1^st and 3^rd week postpartum. Higher (P<0.05) ALB level at 6^th week postpartum was observed in GP-III than GP-I. BUN level was significantly (P<0.05) lower in GP-III and GP-II than control. It was lowest (P<0.05) in GP-III at 1st and 3rd week postpartum. Similarly, lower (P<0.05) Cr level was observed in GP-III and II than control and lowest concentration (P<0.05) in GP-III animals.

RPM and RPC supplementation provides energy via hepatic β-oxidation of NEFA sparing glucose in circulation and also stimulates hepatic gluconeogenesis during transition period in dairy cows (Zhou et al., 2016). Higher TP level in both treatment groups can be attributed to protein anabolic effects of RPM and RPC during transition period. Being inflammatory and hepatic biomarker, lower levels of ALB in control suggests hepatic damage during transition (Osorio et al., 2014) thereby implying anti-inflammatory responses of RPM and RPC. This was corroborated by Batisel et al., (2017b) and Zhou et al., (2016). BUN concentration indicates excretion and utilization efficiency of nitrogen in dairy animals (Sun et al., 2016). Choline spares methionine for protein synthesis by donating -CH₃. This might explain decreased BUN due to RPM and RPC supplementation. Creatinine indicates body muscle mass and its catabolism during transition. High BCS with lower muscle mass mobilization during transition is associated with lower Cr (Osorio et al., 2014). In present study lowered Cr and higher BCS in GP-III and II than GP-I indicated lowered muscle mobilization due to RPM and RPC.
 
Liver enzymes
 
Lowest (P<0.05) postpartum AST level was in GP-III. GP-II had lower (P<0.05) AST level at 1^st and 3^rd week postpartum than GP-I. Similarly, lowest (P<0.05) ALT concentration was at 1^st and 3^rd week post-partum in GP-III followed by GP-II and highest (P<0.05) was in GP-I. GGT and GLDH levels were significantly (P<0.05) lowered in GP-III and II during 1^st and 3^rd week postpartum than GP-I.

Around calving AST, ALT, GGT and GLDH are key liver health biomarkers (Osorio et al., 2014) and their high concentration indicates hepatic damage (Batisel et al., 2017a). Results of present study imply improvement of liver health by RPM and RPC supplementation.
 
Hormones
 
Amongst hormones GP-III had significantly (P<0.05) higher T₃ level at 1^st and 6^th week postpartum than control. Highest (P<0.05) thyroxine (T₄) level was observed in GP-III followed by II and I during 6^th week postpartum period. Cortisol level was significantly (P<0.05) lower in GP-III and I at 1^st and 6^th week postpartum than control.

Thyroid hormones play metabolic roles and their postpartum reduction reflects decreased secretion rate due to energy deficiency and high demand for these hormones by mammary tissue (Fiore et al., 2018). Similar to findings of Fiore et al., (2018) and Steinhoff et al., (2019) we observed significantly (P<0.05) lower T₃ level in control during 1^st and 6^th week postpartum while T₄ level was lower only at 6thweek postpartum. This might be due to adaptive change for NEB at calving in 1^st week postpartum as well as during increased metabolic demand of approaching peak lactation at 6^th week postpartum. Higher (P<0.05) level of T₃ in GP-III and of T₄ in GP-III and II might be attributed to alleviation of NEB by RPM and RPC supplementation. Conversion of T₄ to T₃ may explain non-significant T₄ differences at 1^st week postpartum.

In the present study, significantly (P<0.05) higher cortisol concentration in control at 1^st and 6^th week postpartum indicates stress without RPM and RPC supplementation.
 
Macro and micro elements
 
Results show highest (P<0.05) level of Ca, P and Mg at 1^st and 3^rd week postpartum in GP-III followed by II and I. Higher pre-partum calcium levels decreased at 1^st and 3^rd week of parturition probably due to its partitioning for milk. This was also true for P and Mg. P under high energy demand during postpartum is used for ATP formation. Observed lower Ca, P and Mg level in GP-I buffaloes may be due to poor feed intake and NEB because of transitional stress. Thus levels of these elements in supplemented groups indicates improved feed intake.
 
Infrared thermography (IRT)
 
Among surface temperature of different body regions (Fig 1) eye temperature (°C) was significantly lower during 1^st week postpartum in GP-III and II than I. More efficient cattle have lower surface temperature due to lower heat loss and methane production (Montanholi et al., 2008).



Possibility of increase in body temperature due to supplementation of RPM and RPC was explored. Only eye temperature was significantly (P<0.05) low in GP-II and III during 1^st week postpartum period. Montanholi et al., (2008) also found significant difference in eye temperature in beef steers with distinct feed efficiencies.

RPM and RPC supplementation increased BCS, blood glucose, TP, ALB, T₃, T₄, Ca, P and Mg levels and decreased BUN, Cr, AST, ALT, GGT, GLDH and cortisol levels during transition period in Surti buffaloes. RPM and RPC supplementation also decreased RT, TT and ET at 1^st week postpartum period in Surti buffaloes. Supplementation of RPM +RPC as compared to RPM alone, gave better results in terms of biochemical analytes in transition Surti buffaloes.