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Full Research Article

Impact of Nutritional Supplementation during the Peri-partum Period on Negative Energy Balance Indicators (Nefa and Bhba) and Biochemical Parameters in Gir Cows

B
B. Sahoo1
M
M.T. Panchal1
B
B.P. Chavda1
J
J.A. Patel1
D
D.K. Patel1
S
S.S. Biswal2,*
1Department of Gynaecology and Obstetrics, Veterinary College, Kamdhenu University, Anand-388 001, Gujarat, India.
2Department of Animal Reproduction, Gynecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.

ABSTRACT

Background: The study evaluated the effect of nutritional supplementation during the peripartum period on negative energy balance (NEB) indicators non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) and other biochemical parameters in Gir cows.

Methods: Eighteen advanced pregnant Gir cows were divided into three groups: Group I received bypass fat and a methionine-lysine-fortified mineral mixture; Group II received only the mineral mixture; and Group III served as the control. Blood samples were collected at specific intervals pre- and postpartum to analyze glucose, total proteins, total cholesterol, NEFA and BHBA levels.

Result: It was found that supplemented cows (Groups I and II) had significantly lower plasma NEFA levels (p<0.05) compared to controls, suggesting better adaptation to NEB. Plasma BHBA levels, though not significantly different, declined faster in supplemented cows, indicating improved metabolic recovery. Blood glucose levels peaked at calving and were numerically higher in supplemented cows postpartum. Total protein and cholesterol levels showed no significant variation among groups, though supplemented cows exhibited higher protein levels at 7 and 14 days postpartum.

INTRODUCTION

The postpartum health, production and reproduction of dairy animals largely depend on the nutritional and energy status peripartum (Singh et al., 2016). Peri-parturient period, which is often considered to be between 3 weeks pre-partum to 3 weeks post-partum, is the most stressful phase of transition for dairy cows from pregnant non-lactating state to lactation (Verma et al., 2025). The postpartum health, production and reproduction of dairy animals largely depend on the nutritional and energy status peripartum (Dhami et al., 2026). Energy balance is a critical factor in dairy cattle research, particularly concerning reproductive performance and milk production. Early lactation often sees cows prioritizing energy allocation toward milk production at the expense of reproduction, leading to NEB. The concept of homeorhesis, introduced by Bauman and Currie (1980), explains the physiological prioritization of energy and nutrients during this period. Circulating NEFA and BHBA are key indicators of NEB or ketosis. While some elevation of these metabolites is normal during early lactation as cows balance energy intake and demands, excessive levels indicate inadequate adaptation to NEB (Herdt, 2000).

The transition period, characterized by reduced dry matter intake, leads to a sharp decline in glucose levels and increased mobilization of body fat as NEFA. This results in the accumulation of BHBA, a by-product of incomplete NEFA oxidation (Vazquez-Añon et al., 1994). Nutritional supplementation, particularly bypass fat, during the peripartum period may mitigate the adverse effects of NEB, improving reproductive performance, milk production and lactation persistency. Hence, it is proposed to utilize this knowledge by including macro-micronutrients and bypass fat in the diet during the transition period to evaluate changes in biochemical parameters in both treated and control Gir cows. 

MATERIALS AND METHODS

The study involved eighteen advanced pregnant Gir cows of second to fourth parity with comparable body sizes. Advanced pregnancy was confirmed through breeding records and gynaecological examinations. Approximately one month before delivery, the cows were randomly assigned to control and supplement groups. All animals calved without complications.

The cows were divided into three groups of six each: one control group and two treatment groups. Group I received a mineral mixture containing rumen-protected methionine and lysine (50 g/day/head) for 4 weeks prepartum and 60 days postpartum, along with bypass fat (100 g/day) for 4 weeks prepartum and 2 weeks postpartum. Postpartum bypass fat was adjusted based on milk yield (10 g/liter), capped at 150g/day until 60 days postpartum.  Group II received the same mineral mixture for 30 days prepartum and 60 days postpartum. Group III (Control) followed the routine farm feeding schedule (concentrates and grazing) without supplements.

Blood samples (8-10 ml) were collected from the jugular vein at 28, 14 and 7 days prepartum, on the day of calving (0 day) and at 7, 14, 28, 42 and 60 days postpartum. Plasma was separated via centrifugation (3000 rpm for 10 minutes) and stored at -20°C in sterilized vials with merthiolate as a preservative. Plasma samples were analyzed for metabolic and hormonal parameters using standard procedures and auto-analyzers.
 
Blood glucose
 
Analyzed using the Accu-Check Integra Kit (Roche Diagnostics India Pvt. Ltd., Mumbai) via the direct strip method. A drop of freshly collected blood was applied to the strip and results were displayed automatically.
 
Total proteins
 
Estimated using the Biuret method (Doumas et al., 1975) with assay kits from Crest Biosystems and a Mindray Chemistry Analyzer BS-120.
 
Total cholesterol
 
Measured using the CHOD/PAP method (Allain et al., 1974) with assay kits from Crest Biosystems and a Mindray Chemistry Analyzer BS-120.

The estimation of non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) was conducted using ELISA assay kits (KRISHGEN BioSystems, INDIA) following standard procedures. For both assays, 50 µl of standard solution was added to standard wells, while 40 µl of sample and 10 µl of biotinylated antibody (NEFA or BHBA) were added to sample wells. The reaction was initiated by adding 50 µl of Streptavidin: HRP conjugate, followed by incubation at 37°C in the dark for 60 minutes. After washing the plate with diluted Wash Buffer (1X), 50 µl each of TMB substrate A and B were added and the plate was incubated at 37°C for 10 minutes. The reaction was stopped by adding 50 µl of Stop Solution and absorbance was measured at 450 nm using a Multiskan™ FC Microplate Photometer (Thermo Scientific). A standard curve was plotted using GraphPad Prism 9.0 software, with mean absorbance (450 nm) on the Y-axis and corresponding concentrations on the X-axis, to determine the concentrations of NEFA and BHBA in the samples.The standard curve was drawn by software (Graphpad Prism 9.0) and automated results were obtained for the unknown NEFA and BHBA concentrations.

The data obtained on the above plasma biochemical profiles were analyzed using ANOVA (Analysis of Variance) followed by the Post-hoc Duncan test with SPSS 20.0 software.

RESULTS AND DISCUSSION

The mean blood glucose (mg/dl) concentrations in Gir cows supplemented with bypass fat and mineral mixture (Group I) increased significantly (p<0.05) from 7 days prepartum (68.17±2.30) to peak at parturition (78.17±3.96), then returned to prepartum levels by 7 days postpartum and fluctuated non-significantly until 60 days postpartum (Table 1). Similar trends were observed in cows receiving only mineral mixture (Group II) and the control group (Group III), with peak values at parturition (74.17±2.27 and 70.17±1.74, respectively), followed by a rapid decline by 7 days postpartum and non-significant fluctuations thereafter. The overall mean pooled blood glucose values across all groups ranged from 57.39±3.16 to 74.17±1.72 mg/dl. No significant differences were found between Group I, II and III. However, day-wise postpartum observations showed non-significantly higher blood glucose levels in Group I compared to Groups II and III.

Table 1: Mean blood glucose (mg/dl) and total protein levels (g/dl) levels during peripartum periods in Gir cows under nutrient supplemented (treatment) and control groups.



The mean plasma total protein concentrations in Gir cows under the control group (III) were significantly higher (p<0.05) prepartum and at calving, declined by 7 days postpartum and recovered by 60 days postpartum (Table 1). Treated cows showed no significant trends. Plasma total protein levels were significantly higher (p<0.05) in Group I (11.19±0.35, 11.68±0.55 g/dl) compared to Group II (9.67±0.37, 9.84±0.33 g/dl) and Group III (9.67±0.37, 9.84±0.33 g/dl) on 7 and 14 days postpartum. Pooled mean values across groups ranged from 10.17±0.27 to 10.82±0.24 g/dl.

The mean plasma total cholesterol concentrations in cows supplemented with methionine- and lysine-fortified mineral mixture (Group II) declined prepartum, reaching the lowest value (p<0.01) at calving, followed by a non-significant increase until 14 days postpartum and a significant rise (p<0.01) thereafter, peaking at 60 days postpartum (Table 2). Groups I and III showed similar trends, with non-significant differences. Overall, cholesterol levels decreased prepartum, rose gradually postpartum and peaked at 60 days, with pooled values ranging from 111.13±7.83 to 188.28±8.76 mg/dl. Although bypass fat and mineral mixture supplementation (Group I) did not significantly affect cholesterol levels, Group I cows exhibited apparently higher levels compared to Groups II and III, suggesting a potential influence of bypass fat on cholesterol metabolism.

Table 2: Mean plasma total cholesterol (mg/dl) levels during peripartum periods in Gir cows under nutrient supplemented (treatment) and control groups.



The mean plasma NEFA concentrations (nmol/ml) in Gir cows supplemented with bypass fat and mineral mixture fortified with methionine and lysine (Group I) increased significantly (p<0.05) from 7 days prepartum (60.68±13.18) to peak at calving (108.67±12.18), remaining high until 7 days postpartum (107.52±7.18), before declining by 14 days postpartum (91.67±11.71) and fluctuating non-significantly until 60 days postpartum (Table 3). Similarly, Group II cows showed a significant rise (p<0.05) in NEFA levels, peaking at calving (135.34±24.38) and remaining elevated until 7 days postpartum (128.60±25.10), followed by a non-significant decline. Control cows (Group III) exhibited no significant variations, though the highest NEFA levels (174.96±21.37) were observed at calving, with a slight postpartum decline. Overall, NEFA concentrations in Groups I and II were significantly lower (p<0.05) than in Group III during both pre-and postpartum periods.

Table 3: Mean plasma NEFA (nmol/ml) and BHBA(µg/ml) levels during peripartumperiod in Gir cows under nutrient supplemented (treatment) and control groups.



The mean plasma BHBA concentrations (µg/ml) in Gir cows supplemented with bypass fat and mineral mixture fortified with methionine and lysine (Group I) increased significantly (p<0.01) from 7 days prepartum (86.52±8.25) to peak at calving (122.19±7.57), remaining elevated until 7 days postpartum (111.18±7.57), before declining by 14 days postpartum (99.54±6.24) and fluctuating non-significantly until 60 days postpartum animals (Table 3). Group II cows showed a similar trend, with BHBA levels rising significantly (p<0.05) to peak at calving (124.23±5.51) and declining non-significantly thereafter. Control cows (Group III) also peaked at calving (133.61±7.49, p<0.01) but took longer to decline, reaching prepartum levels only by 28 days postpartum (85.71±7.15).

While nutrient supplementation did not significantly affect BHBA levels compared to controls, treated cows (Groups I and II) recovered from elevated BHBA levels by 14 days postpartum, whereas animals in control group took until 28 days. This suggests that bypass fat and mineral mixture supplementation may help cows recover more quickly from negative energy balance, highlighting their potential role in improving metabolic adaptation during the peripartum period.

The mean blood glucose trends in Gir cows across all groups align with findings by Kalasariya et al., (2017). Pooled blood glucose values were comparable to those reported by Anam et al. (2021). Blood glucose levels reflect carbohydrate status and while hypoglycaemic herds may not show clinical symptoms, anoestrus remains a concern, particularly in winter (King, 1971).

In this study, blood glucose differences between supplemented (Groups I and II) and control (Group III) cows were non-significant, consistent with Chavda et al., (2022a) and Karimi et al., (2023). However, Vala et al., (2020) reported significant improvements with nutritional supplementation. Postpartum, Group I cows showed non-significantly higher glucose levels, suggesting bypass fat may help mitigate negative energy balance. Butler et al., (2000) linked low glucose levels to delayed ovulation due to inhibited LH pulses, explaining the 10-day delay in postpartum oestrus in control cows, likely due to poorer energy balance adaptation.

The mean plasma total protein concentrations in control Gir cows (Group III) were significantly higher (p<0.05) prepartum and at calving, declined by 7 days postpartum and recovered by 60 days postpartum. Similar trends were reported by Setia et al., (1992), while Sallam et al., (2021) noted an increase from day 10 to 30 postpartum, followed by a decline. Protein deficiency, linked to delayed postpartum oestrus (Roberts, 1971), may explain the delayed oestrus in control cows.

Treated cows showed no significant trends in plasma total protein, consistent with Vala et al., (2020). However, Group I cows had significantly higher (p<0.05) protein levels on 7 and 14 days postpartum compared to Groups II and III, aligning with findings by Bicalho et al., (2014). Pooled mean protein values in this study were comparable to Nasiri et al. (2022), though lower values were reported by Vala et al., (2020). These results suggest that bypass fat and mineral supplementation may support better protein metabolism postpartum.

The mean plasma total cholesterol levels in this study decreased prepartum, reaching the lowest at calving and gradually increased postpartum, peaking at 60 days. This aligns with findings by Chavda et al. (2022). Lower cholesterol around parturition may result from estrogen and thyroid hormones inhibiting cholesterogenesis (Prakash and Tandon, 1979), while postpartum increases are linked to upregulated cholesterol biosynthesis for lactation (Kessler et al., 2014). Cholesterol, a steroid hormone precursor, is closely tied to reproductive physiology, supporting follicular activity and steroidogenesis (Robinson, 1977). Pooled cholesterol values were comparable to Dhami et al. (2017), but differed from Kalasariya et al., (2017). Group I cows (bypass fat and mineral mixture) showed higher cholesterol levels than Groups II and III, which is in agreement with Tyagi et al., (2010) and Ranjan et al., (2012), who reported significant effects of bypass fat on cholesterol. Dietary fat increases cholesterol and progesterone, enhancing luteal function (Staples and Thatcher, 2005), suggesting the role of bypass fat in improving reproductive outcomes. The discussion suggests that rising plasma total cholesterol levels may support follicular development and steroidogenesis in cows, particularly in supplemented groups. Cholesterol likely serves as a precursor for reproductive steroid hormones and its increased levels during lactation may be a physiological adaptation to meet lactation demands.

The lower plasma mean NEFA levels before parturition and postpartum period in both the treatment groups (I and II) might be due to the positive effect of supplementation of bypass fat and mineral mixture. However, Staples et al. (1998) opined that dietary fat decreases lipogenesis by adipose tissues, resulting in enhanced lipolysis and higher NEFA readings. In this study, Gir cows in Groups I and II showed significantly (p<0.05) lower mean plasma NEFA concentrations compared to Group III (control) during prepartum and postpartum periods. These findings are in agreement with Vala et al., (2020), who observed reduced NEFA levels in supplemented Jaffarabadi buffaloes. However, contrasting results were reported by Sallam et al., (2021), who found no significant effect of nutrient supplementation on plasma NEFA levels in dairy cows. Sutaria et al., (2025) reported no significant difference of NEFA levels at different time point postpartum in Mehsana Buffaloes. The discrepancy may stem from variations in supplementation type, dosage, or animal breed.

In the present study, the animals under control group required longer period to recover from the state of negative energy balance, indicative of positive response of treated cows with bypass fat and/or mineral mixture supplementation. The present findings of the trend observed for mean plasma BHBA levels are in accordance with the reports of Theodore et al., (2017); Chavda et al. (2022), who observed no significance effect of the nutrient supplementation on mean plasma BHBA levels of the treatment group as compared to the control in HF crossbred and Gir cows, respectively. The trend of the pooled mean plasma NEFA and BHBA in Gir cows observed in the present study corroborated well with the observations of Garverick et al. (2013) and Sutaria et al., (2025).

The transition period involves reduced dry matter intake, leading to lower glucose levels and increased fat mobilization as NEFA, resulting in by-products like BHBA. While slight rises in NEFA and BHBA are normal during early lactation, excessive levels indicate poor adaptation to negative energy balance (NEB), signalling ketosis risk (Vazquez-Anon et al., 1994; Herdt, 2000).

CONCLUSION

The mean plasma total protein levels did not show any variation among treated and untreated Gir cows due to supplementation of bypass fat and mineral mixture together or alone.Blood glucose levels were numerically higher in supplemented cows postpartum, peaking at calving, indicating a maternal physiological response to increased galactopoiesis demands. Plasma total cholesterol levels declined prepartum, rose gradually postpartum and peaked at 60 days. Peripartum supplementation of bypass fat and mineral mixture fortified with methionine and lysine together or alone in Gir cows reduced the plasma NEFA and BHBA levels in the treated cows, indicative of better acclimatization to negative energy balance of treated cows and supports better postpartum recovery in Gir cows.

ACKNOWLEDGEMENT

We extend our gratitude to the authorities of Kamdhenu University (KU) and Goraj Muniseva Ashram, Waghodiya, for their invaluable support and cooperation throughout this study. We also acknowledge the financial assistance provided by ICAR, New Delhi and NIANP, Bangalore, under the sanctioned project.
 
Informed consent
 
All animal procedures and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare no conflicts of interest regarding this article’s publication. No funding influenced the study design, data collection, analysis, publication decision, or manuscript preparation.

REFERENCES


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    Full Research Article

    Impact of Nutritional Supplementation during the Peri-partum Period on Negative Energy Balance Indicators (Nefa and Bhba) and Biochemical Parameters in Gir Cows

    B
    B. Sahoo1
    M
    M.T. Panchal1
    B
    B.P. Chavda1
    J
    J.A. Patel1
    D
    D.K. Patel1
    S
    S.S. Biswal2,*
    1Department of Gynaecology and Obstetrics, Veterinary College, Kamdhenu University, Anand-388 001, Gujarat, India.
    2Department of Animal Reproduction, Gynecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.

    ABSTRACT

    Background: The study evaluated the effect of nutritional supplementation during the peripartum period on negative energy balance (NEB) indicators non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) and other biochemical parameters in Gir cows.

    Methods: Eighteen advanced pregnant Gir cows were divided into three groups: Group I received bypass fat and a methionine-lysine-fortified mineral mixture; Group II received only the mineral mixture; and Group III served as the control. Blood samples were collected at specific intervals pre- and postpartum to analyze glucose, total proteins, total cholesterol, NEFA and BHBA levels.

    Result: It was found that supplemented cows (Groups I and II) had significantly lower plasma NEFA levels (p<0.05) compared to controls, suggesting better adaptation to NEB. Plasma BHBA levels, though not significantly different, declined faster in supplemented cows, indicating improved metabolic recovery. Blood glucose levels peaked at calving and were numerically higher in supplemented cows postpartum. Total protein and cholesterol levels showed no significant variation among groups, though supplemented cows exhibited higher protein levels at 7 and 14 days postpartum.

    INTRODUCTION

    The postpartum health, production and reproduction of dairy animals largely depend on the nutritional and energy status peripartum (Singh et al., 2016). Peri-parturient period, which is often considered to be between 3 weeks pre-partum to 3 weeks post-partum, is the most stressful phase of transition for dairy cows from pregnant non-lactating state to lactation (Verma et al., 2025). The postpartum health, production and reproduction of dairy animals largely depend on the nutritional and energy status peripartum (Dhami et al., 2026). Energy balance is a critical factor in dairy cattle research, particularly concerning reproductive performance and milk production. Early lactation often sees cows prioritizing energy allocation toward milk production at the expense of reproduction, leading to NEB. The concept of homeorhesis, introduced by Bauman and Currie (1980), explains the physiological prioritization of energy and nutrients during this period. Circulating NEFA and BHBA are key indicators of NEB or ketosis. While some elevation of these metabolites is normal during early lactation as cows balance energy intake and demands, excessive levels indicate inadequate adaptation to NEB (Herdt, 2000).

    The transition period, characterized by reduced dry matter intake, leads to a sharp decline in glucose levels and increased mobilization of body fat as NEFA. This results in the accumulation of BHBA, a by-product of incomplete NEFA oxidation (Vazquez-Añon et al., 1994). Nutritional supplementation, particularly bypass fat, during the peripartum period may mitigate the adverse effects of NEB, improving reproductive performance, milk production and lactation persistency. Hence, it is proposed to utilize this knowledge by including macro-micronutrients and bypass fat in the diet during the transition period to evaluate changes in biochemical parameters in both treated and control Gir cows. 

    MATERIALS AND METHODS

    The study involved eighteen advanced pregnant Gir cows of second to fourth parity with comparable body sizes. Advanced pregnancy was confirmed through breeding records and gynaecological examinations. Approximately one month before delivery, the cows were randomly assigned to control and supplement groups. All animals calved without complications.

    The cows were divided into three groups of six each: one control group and two treatment groups. Group I received a mineral mixture containing rumen-protected methionine and lysine (50 g/day/head) for 4 weeks prepartum and 60 days postpartum, along with bypass fat (100 g/day) for 4 weeks prepartum and 2 weeks postpartum. Postpartum bypass fat was adjusted based on milk yield (10 g/liter), capped at 150g/day until 60 days postpartum.  Group II received the same mineral mixture for 30 days prepartum and 60 days postpartum. Group III (Control) followed the routine farm feeding schedule (concentrates and grazing) without supplements.

    Blood samples (8-10 ml) were collected from the jugular vein at 28, 14 and 7 days prepartum, on the day of calving (0 day) and at 7, 14, 28, 42 and 60 days postpartum. Plasma was separated via centrifugation (3000 rpm for 10 minutes) and stored at -20°C in sterilized vials with merthiolate as a preservative. Plasma samples were analyzed for metabolic and hormonal parameters using standard procedures and auto-analyzers.
     
    Blood glucose
     
    Analyzed using the Accu-Check Integra Kit (Roche Diagnostics India Pvt. Ltd., Mumbai) via the direct strip method. A drop of freshly collected blood was applied to the strip and results were displayed automatically.
     
    Total proteins
     
    Estimated using the Biuret method (Doumas et al., 1975) with assay kits from Crest Biosystems and a Mindray Chemistry Analyzer BS-120.
     
    Total cholesterol
     
    Measured using the CHOD/PAP method (Allain et al., 1974) with assay kits from Crest Biosystems and a Mindray Chemistry Analyzer BS-120.

    The estimation of non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) was conducted using ELISA assay kits (KRISHGEN BioSystems, INDIA) following standard procedures. For both assays, 50 µl of standard solution was added to standard wells, while 40 µl of sample and 10 µl of biotinylated antibody (NEFA or BHBA) were added to sample wells. The reaction was initiated by adding 50 µl of Streptavidin: HRP conjugate, followed by incubation at 37°C in the dark for 60 minutes. After washing the plate with diluted Wash Buffer (1X), 50 µl each of TMB substrate A and B were added and the plate was incubated at 37°C for 10 minutes. The reaction was stopped by adding 50 µl of Stop Solution and absorbance was measured at 450 nm using a Multiskan™ FC Microplate Photometer (Thermo Scientific). A standard curve was plotted using GraphPad Prism 9.0 software, with mean absorbance (450 nm) on the Y-axis and corresponding concentrations on the X-axis, to determine the concentrations of NEFA and BHBA in the samples.The standard curve was drawn by software (Graphpad Prism 9.0) and automated results were obtained for the unknown NEFA and BHBA concentrations.

    The data obtained on the above plasma biochemical profiles were analyzed using ANOVA (Analysis of Variance) followed by the Post-hoc Duncan test with SPSS 20.0 software.

    RESULTS AND DISCUSSION

    The mean blood glucose (mg/dl) concentrations in Gir cows supplemented with bypass fat and mineral mixture (Group I) increased significantly (p<0.05) from 7 days prepartum (68.17±2.30) to peak at parturition (78.17±3.96), then returned to prepartum levels by 7 days postpartum and fluctuated non-significantly until 60 days postpartum (Table 1). Similar trends were observed in cows receiving only mineral mixture (Group II) and the control group (Group III), with peak values at parturition (74.17±2.27 and 70.17±1.74, respectively), followed by a rapid decline by 7 days postpartum and non-significant fluctuations thereafter. The overall mean pooled blood glucose values across all groups ranged from 57.39±3.16 to 74.17±1.72 mg/dl. No significant differences were found between Group I, II and III. However, day-wise postpartum observations showed non-significantly higher blood glucose levels in Group I compared to Groups II and III.

    Table 1: Mean blood glucose (mg/dl) and total protein levels (g/dl) levels during peripartum periods in Gir cows under nutrient supplemented (treatment) and control groups.



    The mean plasma total protein concentrations in Gir cows under the control group (III) were significantly higher (p<0.05) prepartum and at calving, declined by 7 days postpartum and recovered by 60 days postpartum (Table 1). Treated cows showed no significant trends. Plasma total protein levels were significantly higher (p<0.05) in Group I (11.19±0.35, 11.68±0.55 g/dl) compared to Group II (9.67±0.37, 9.84±0.33 g/dl) and Group III (9.67±0.37, 9.84±0.33 g/dl) on 7 and 14 days postpartum. Pooled mean values across groups ranged from 10.17±0.27 to 10.82±0.24 g/dl.

    The mean plasma total cholesterol concentrations in cows supplemented with methionine- and lysine-fortified mineral mixture (Group II) declined prepartum, reaching the lowest value (p<0.01) at calving, followed by a non-significant increase until 14 days postpartum and a significant rise (p<0.01) thereafter, peaking at 60 days postpartum (Table 2). Groups I and III showed similar trends, with non-significant differences. Overall, cholesterol levels decreased prepartum, rose gradually postpartum and peaked at 60 days, with pooled values ranging from 111.13±7.83 to 188.28±8.76 mg/dl. Although bypass fat and mineral mixture supplementation (Group I) did not significantly affect cholesterol levels, Group I cows exhibited apparently higher levels compared to Groups II and III, suggesting a potential influence of bypass fat on cholesterol metabolism.

    Table 2: Mean plasma total cholesterol (mg/dl) levels during peripartum periods in Gir cows under nutrient supplemented (treatment) and control groups.



    The mean plasma NEFA concentrations (nmol/ml) in Gir cows supplemented with bypass fat and mineral mixture fortified with methionine and lysine (Group I) increased significantly (p<0.05) from 7 days prepartum (60.68±13.18) to peak at calving (108.67±12.18), remaining high until 7 days postpartum (107.52±7.18), before declining by 14 days postpartum (91.67±11.71) and fluctuating non-significantly until 60 days postpartum (Table 3). Similarly, Group II cows showed a significant rise (p<0.05) in NEFA levels, peaking at calving (135.34±24.38) and remaining elevated until 7 days postpartum (128.60±25.10), followed by a non-significant decline. Control cows (Group III) exhibited no significant variations, though the highest NEFA levels (174.96±21.37) were observed at calving, with a slight postpartum decline. Overall, NEFA concentrations in Groups I and II were significantly lower (p<0.05) than in Group III during both pre-and postpartum periods.

    Table 3: Mean plasma NEFA (nmol/ml) and BHBA(µg/ml) levels during peripartumperiod in Gir cows under nutrient supplemented (treatment) and control groups.



    The mean plasma BHBA concentrations (µg/ml) in Gir cows supplemented with bypass fat and mineral mixture fortified with methionine and lysine (Group I) increased significantly (p<0.01) from 7 days prepartum (86.52±8.25) to peak at calving (122.19±7.57), remaining elevated until 7 days postpartum (111.18±7.57), before declining by 14 days postpartum (99.54±6.24) and fluctuating non-significantly until 60 days postpartum animals (Table 3). Group II cows showed a similar trend, with BHBA levels rising significantly (p<0.05) to peak at calving (124.23±5.51) and declining non-significantly thereafter. Control cows (Group III) also peaked at calving (133.61±7.49, p<0.01) but took longer to decline, reaching prepartum levels only by 28 days postpartum (85.71±7.15).

    While nutrient supplementation did not significantly affect BHBA levels compared to controls, treated cows (Groups I and II) recovered from elevated BHBA levels by 14 days postpartum, whereas animals in control group took until 28 days. This suggests that bypass fat and mineral mixture supplementation may help cows recover more quickly from negative energy balance, highlighting their potential role in improving metabolic adaptation during the peripartum period.

    The mean blood glucose trends in Gir cows across all groups align with findings by Kalasariya et al., (2017). Pooled blood glucose values were comparable to those reported by Anam et al. (2021). Blood glucose levels reflect carbohydrate status and while hypoglycaemic herds may not show clinical symptoms, anoestrus remains a concern, particularly in winter (King, 1971).

    In this study, blood glucose differences between supplemented (Groups I and II) and control (Group III) cows were non-significant, consistent with Chavda et al., (2022a) and Karimi et al., (2023). However, Vala et al., (2020) reported significant improvements with nutritional supplementation. Postpartum, Group I cows showed non-significantly higher glucose levels, suggesting bypass fat may help mitigate negative energy balance. Butler et al., (2000) linked low glucose levels to delayed ovulation due to inhibited LH pulses, explaining the 10-day delay in postpartum oestrus in control cows, likely due to poorer energy balance adaptation.

    The mean plasma total protein concentrations in control Gir cows (Group III) were significantly higher (p<0.05) prepartum and at calving, declined by 7 days postpartum and recovered by 60 days postpartum. Similar trends were reported by Setia et al., (1992), while Sallam et al., (2021) noted an increase from day 10 to 30 postpartum, followed by a decline. Protein deficiency, linked to delayed postpartum oestrus (Roberts, 1971), may explain the delayed oestrus in control cows.

    Treated cows showed no significant trends in plasma total protein, consistent with Vala et al., (2020). However, Group I cows had significantly higher (p<0.05) protein levels on 7 and 14 days postpartum compared to Groups II and III, aligning with findings by Bicalho et al., (2014). Pooled mean protein values in this study were comparable to Nasiri et al. (2022), though lower values were reported by Vala et al., (2020). These results suggest that bypass fat and mineral supplementation may support better protein metabolism postpartum.

    The mean plasma total cholesterol levels in this study decreased prepartum, reaching the lowest at calving and gradually increased postpartum, peaking at 60 days. This aligns with findings by Chavda et al. (2022). Lower cholesterol around parturition may result from estrogen and thyroid hormones inhibiting cholesterogenesis (Prakash and Tandon, 1979), while postpartum increases are linked to upregulated cholesterol biosynthesis for lactation (Kessler et al., 2014). Cholesterol, a steroid hormone precursor, is closely tied to reproductive physiology, supporting follicular activity and steroidogenesis (Robinson, 1977). Pooled cholesterol values were comparable to Dhami et al. (2017), but differed from Kalasariya et al., (2017). Group I cows (bypass fat and mineral mixture) showed higher cholesterol levels than Groups II and III, which is in agreement with Tyagi et al., (2010) and Ranjan et al., (2012), who reported significant effects of bypass fat on cholesterol. Dietary fat increases cholesterol and progesterone, enhancing luteal function (Staples and Thatcher, 2005), suggesting the role of bypass fat in improving reproductive outcomes. The discussion suggests that rising plasma total cholesterol levels may support follicular development and steroidogenesis in cows, particularly in supplemented groups. Cholesterol likely serves as a precursor for reproductive steroid hormones and its increased levels during lactation may be a physiological adaptation to meet lactation demands.

    The lower plasma mean NEFA levels before parturition and postpartum period in both the treatment groups (I and II) might be due to the positive effect of supplementation of bypass fat and mineral mixture. However, Staples et al. (1998) opined that dietary fat decreases lipogenesis by adipose tissues, resulting in enhanced lipolysis and higher NEFA readings. In this study, Gir cows in Groups I and II showed significantly (p<0.05) lower mean plasma NEFA concentrations compared to Group III (control) during prepartum and postpartum periods. These findings are in agreement with Vala et al., (2020), who observed reduced NEFA levels in supplemented Jaffarabadi buffaloes. However, contrasting results were reported by Sallam et al., (2021), who found no significant effect of nutrient supplementation on plasma NEFA levels in dairy cows. Sutaria et al., (2025) reported no significant difference of NEFA levels at different time point postpartum in Mehsana Buffaloes. The discrepancy may stem from variations in supplementation type, dosage, or animal breed.

    In the present study, the animals under control group required longer period to recover from the state of negative energy balance, indicative of positive response of treated cows with bypass fat and/or mineral mixture supplementation. The present findings of the trend observed for mean plasma BHBA levels are in accordance with the reports of Theodore et al., (2017); Chavda et al. (2022), who observed no significance effect of the nutrient supplementation on mean plasma BHBA levels of the treatment group as compared to the control in HF crossbred and Gir cows, respectively. The trend of the pooled mean plasma NEFA and BHBA in Gir cows observed in the present study corroborated well with the observations of Garverick et al. (2013) and Sutaria et al., (2025).

    The transition period involves reduced dry matter intake, leading to lower glucose levels and increased fat mobilization as NEFA, resulting in by-products like BHBA. While slight rises in NEFA and BHBA are normal during early lactation, excessive levels indicate poor adaptation to negative energy balance (NEB), signalling ketosis risk (Vazquez-Anon et al., 1994; Herdt, 2000).

    CONCLUSION

    The mean plasma total protein levels did not show any variation among treated and untreated Gir cows due to supplementation of bypass fat and mineral mixture together or alone.Blood glucose levels were numerically higher in supplemented cows postpartum, peaking at calving, indicating a maternal physiological response to increased galactopoiesis demands. Plasma total cholesterol levels declined prepartum, rose gradually postpartum and peaked at 60 days. Peripartum supplementation of bypass fat and mineral mixture fortified with methionine and lysine together or alone in Gir cows reduced the plasma NEFA and BHBA levels in the treated cows, indicative of better acclimatization to negative energy balance of treated cows and supports better postpartum recovery in Gir cows.

    ACKNOWLEDGEMENT

    We extend our gratitude to the authorities of Kamdhenu University (KU) and Goraj Muniseva Ashram, Waghodiya, for their invaluable support and cooperation throughout this study. We also acknowledge the financial assistance provided by ICAR, New Delhi and NIANP, Bangalore, under the sanctioned project.
     
    Informed consent
     
    All animal procedures and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare no conflicts of interest regarding this article’s publication. No funding influenced the study design, data collection, analysis, publication decision, or manuscript preparation.

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