Impact of non-enzymatic antioxidants on the biochemical profile in buffaloes having uterine torsion

Introduction

Buffaloes are considered the backbone of animal husbandry in India, particularly calves, which are seen as the future of livestock wealth. Buffaloes are highly versatile animals with great potential to bring about social and economic transformation in agrarian communities. As of the latest census (DHAD 2024) [9] , the total buffalo population in India stands at 109.85 million, reflecting an increase of approximately 1.1% compared to the previous census. Reproductive disorders can negatively impact the productivity and reproductive health of dairy animals (Fesseha & Ayele 2020) [11] . Dystocia, also known as calving difficulty, occurs when the first or second stage of labour is prolonged and assistance is needed for delivery (Weldeyohanes and Fesseha 2020) [32] . In buffaloes, uterine torsion is the leading cause of dystocia, accounting for 41.3% of cases (Juneja et al., 2023) [16] . Torsion of the uterus in buffaloes typically occurs in the pregnant uterine horn and is defined as the twisting of the uterus along its longitudinal axis. Parturition is a stressful event in the life of an animal as evidenced by changes in certain haematological and biochemical parameters (Joshi et al., 2018). Uterine torsion places additional stress on the bovine body, causing significant biochemical changes that vary depending on the severity and duration of the condition (Tripathi & Mehta 2016) [30] . Oxidative stress occurs due to an imbalance between pro-oxidants and antioxidants, leading to cell damage. This disruption results in the production of reactive oxygen species (ROS) (Gutteridge & Halliwell 2018) [13] . Non-enzymatic antioxidants, such as vitamin E along with selenium and vitamin C, helps in reduction of oxidative stress and improve the blood biochemical profile of buffaloes suffering from uterine torsion. Vitamin E, a fat-soluble vitamin, is a strong antioxidant agent act synergistically with the selenium (Xiao et al., 2021) [34] . Vitamin C is a potent reducing agent and scavenger of free radicals in biological systems (Pehlivan et al., 2017) [23] . This study aimed to improve the biochemical profile by utilizing non-enzymatic antioxidants, Blood sampling schedule A total of three blood samples (10 ml/animal) were taken from each animal at the time of reporting, one hour after detorsion and 24 hours of correction of uterine torsion to access the biochemical profiles. Blood samples collected were than centrifuged at 3000 rpm for 15 minutes to separate serum and were stored at-20 °C until the analysis of biochemical parameters.

Estimation of biochemical parameters-

Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), blood urea nitrogen (BUN), albumin (ALB), globulin (GLB), albumin-to-globulin ratio, creatinine (CR), calcium (Ca) and phosphorus (P) were analysed by a semi-auto analyser (Spectra lab Genie Biochemistry Analyzer) as per the protocols given in the respective kits manufactured by Spectrum Medical Industries Private Limited, India.

Statistical analysis

Data obtained were subjected to analysis by one-way analysis of variance (ANOVA) technique (Snedecor & Cochran 2004) [27] using the SPSS software version 21. To know statistical significance the means of different experimental groups were tested (P<0.05) by Duncan's Multiple Range Test (Duncan, 1955) [10] .

Results and Discussion

The results of the current experiment are presented to define the effects of parenteral administration of vitamin E with Se and vitamin C injections on serum biochemical profiles in uterine torsion affected buffaloes (Table -2).

Effect of administration of antioxidants on serum total proteins, albumin, and globulin-

In the present study, parenteral administration of antioxidants significantly (p<0.05) affected serum total proteins, albumin, and the A/G ratio in buffaloes 24 hours after the correction of uterine torsion. However, no significant effect was observed on serum globulin levels. After 24 hours of correction, intramuscular administration of vitamin E and selenium resulted in the highest serum albumin levels and A/G ratios, followed by vitamin C. These findings align with those of (Helal et al., 2009) [14] , who reported a significant increase in serum total protein levels following oral supplementation of vitamin E and selenium in buffaloes. The lack of significant effect on serum globulin levels in buffalo calves, as observed by (Shinde et al., 2009) [26] , also supports the current findings.

Similarly, (Sushma et al., 2015) [29] did not observe any significant changes in serum globulin levels in ram lambs supplemented with selenium. According to (Mogal et al., 2019) [21] , who found significantly higher (p≤0.01) serum total protein and albumin levels in uterine torsion-affected buffaloes treated with intramuscular vitamin E + Se compared to untreated animals. These findings are also in agreement with (Sultana et al., 2022) [28] , who reported significantly higher serum total protein, albumin levels, and A/G ratios in heat-stressed Holstein Friesian calves supplemented with vitamin E and selenium. Similarly, (Gade et al., 2010) [12] observed a significant increase in A/G ratio in heat-stressed buffaloes supplemented with ascorbic acid compared to the control group. Furthermore, the present study is consistent with previous reports, such as (Youssef et al., 2013) [37] , who recorded significantly higher albumin levels in buffalo bulls treated with vitamin C.

In line with the current findings, (Barwary et al., 2011) [5] reported no significant effect of dietary ascorbic acid supplementation on serum globulin levels in male Meriz kids. Additionally, (Abd-Allah & Zanouny 2014) [1] observed a significant increase in serum total protein levels in heat-stressed ram lambs supplemented with vitamin C during the summer season.

The exact mechanism behind the increased serum total protein in antioxidant-treated animals remains unclear, but it may be attributed to improved protein anabolism and reduced protein catabolism. Vitamin E can boost immunoglobulin production, which may explain the rise in albumin levels. It plays a crucial role in protein synthesis and cellular function. Selenium, in conjunction with vitamin E, helps prevent and repair cellular damage caused by reactive oxygen species (ROS), thereby minimizing oxidative stress. Similarly, vitamin C plays a vital role in amino acid metabolism and protein synthesis, potentially contributing to the increased blood protein concentrations observed in this study. This increase may also be attributed to the ameliorative effects of ascorbic acid in reducing the catabolic activity of stress hormones (Borges et al., 2007) [8] .

Effect of administration of antioxidants on serum enzymatic variables

In this study, the parenteral administration of antioxidants had a significant (p≤0.05) effect on serum ALT and AST levels 24 hours after the correction of uterine torsion in buffaloes. Both antioxidant treatments resulted in significantly lower serum ALT and AST levels compared to the control group. Previous research supports these findings. For instance, (Mahmoud et al., 2014) [20] demonstrated that intramuscular administration of antioxidants, and (Alhidary et al., 2015) [3] showed that oral supplementation with vitamin E and selenium led to a significant reduction in serum ALT and AST levels in sheep. These results are consistent with those of (Yenilmaz et al., 2022) [36] , who observed a substantial decrease in ALT levels (p≤0.05) after intramuscular vitamin E administration in cattle, which helped maintain ALT levels and mitigate liver damage caused by oxidative stress during calving. Similarly, (Bhardwaj et al., 2022) reported a significant reduction (p≤0.05) in serum ALT and AST levels following oral supplementation of vitamin E and selenium in buffaloes exposed to heavy metals. Moreover, the results are aligned with previous studies such as those by (Kassab & Mohammed, 2014) [18] , who found a significant decrease (p≤0.01) in serum ALT and AST levels after oral administration of ascorbic acid in sheep. Hepatic cells play a crucial role in various metabolic processes and contain enzymes like ALT and AST, which are widely used as biomarkers for liver damage and the effectiveness of hepato-protective therapies (Rosser & Gores, 1995) [24] . The administration of antioxidants like vitamin E, vitamin C, and selenium helps preserve liver integrity by protecting it from oxidative stress and supporting its metabolic functions.

Effect of administration of antioxidants on serum metabolites-

In the current study, parenteral administration of vitamin E + Se and vitamin C significantly (p≤0.05) reduced serum creatinine and BUN levels 24 hours after the correction of uterine torsion. These findings are in line with previous research, such as the study by (Kassab & Mohammed, 2014) [18] , which reported a significant reduction in serum creatinine levels in transportation-stressed rams treated with ascorbic acid. Likewise, (Khan et al., 2015) [19] observed the beneficial effects of vitamin E and mineral supplementation in reducing plasma BUN levels in pre-partum buffaloes compared to the non-supplemented control group. (Semsirmboon, 2022) [25] , also reported a significant reduction (p≤0.05) in plasma BUN and creatinine levels following high-dose ascorbic acid supplementation in stressed dairy goats under tropical conditions. Elevated BUN and creatinine levels are commonly associated with increased cortisol concentrations, which can trigger the breakdown of proteins and nucleic acids in muscles during stress (Kannan et al., 2002) [17] . Nonenzymatic antioxidants, such as vitamin E, vitamin C, and selenium, may serve as effective agents in protecting tissues from oxidative damage due to their potent free radicalscavenging and antioxidant properties (Yanardag & Ozsoy-Scan, 2007) [35] .

Effect of administration of antioxidants on serum minerals profile-

In the present study, parenteral administration of antioxidants significantly (p≤0.05) affected serum calcium (Ca) and phosphorus (P) levels 24 hours after the correction of uterine torsion in buffaloes. After 24 hours, intramuscular administration of vitamin E and selenium resulted in significantly higher serum Ca and P levels, followed by vitamin C, in comparison to the untreated group. These findings are in line with those of (Khan et al., 2015) [19] , who observed higher plasma Ca and P levels following oral supplementation of vitamin E and a commercial mineral mixture in pre-partum buffaloes. Similar results were reported by (Behera et al., 2020) [6] , who noted significantly increased serum calcium and phosphorus levels 46 days after vitamin C administration in heat-stressed ducks. The present study also aligns with the findings of (Achi et al., 2022) [2] who found a significant increase in milk phosphorus levels in cows supplemented orally with vitamin E and selenium. Vitamin E helps prevent calcium loss caused by increased oxygen consumption and the production of free radicals (White et al., 2001) [33] . A deficiency in vitamin E has also been linked to hypocalcaemia, as it inhibits the conversion of vitamin D to its active metabolites, which may reduce calcium absorption from the intestine (Norazlina et al.,

2004) [22] . Selenium, known for its protective effect against oxidative stress, also plays a vital role as a trace element essential for regulating thyroid hormones, maintaining the redox state of vitamin C, and supporting the function of other molecules (Arthur et al., 1997) [4] . Additionally, the bioconversion of vitamin D3 to its active form, 1,25-(OH)2D3, is enhanced by utilizing ascorbic acid as a cofactor, which in turn elevates plasma concentrations of 1,25-(OH)2D3. This increase boosts serum Ca levels by enhancing the activity of duodenal calcium-binding proteins (Volker & Weiser, 1993) [31] .

Conclusion

The parenteral administration of both non-enzymatic antioxidants can be considered an effective alternative for improving serum total protein, albumin, A/G ratio, Ca, and P levels, while significantly reducing creatinine, BUN, and the serum enzymatic activities of ALT and AST. Overall, the study demonstrated a positive impact of vitamin E with Se and ascorbic acid on serum biochemical parameters in uterine torsion-affected buffaloes.

Acknowledgement

AcknowledgementThe authors would like to express their sincere gratitude to the Dean of the College of Veterinary and Animal Science, Bikaner, and the Head of the Department of Veterinary Gynaecology and Obstetrics, CVAS, Bikaner, Rajasthan, for their financial and technical support. Additionally, the authors would like to express their gratitude to the field veterinarian for providing the essential samples for the research.

References

1. Abd-Allah M, Zanouny AI. Ameliorative effect of administration and chilled drinking water on ram lambs exposed to heat stress during summer season. Egypt J Sheep Goat Sci. 2014;9(1):17-28.
2. Achi NP, Achi JN, Rekwot GZ, Mallam I, Sanni RT, Barje PP. Milk yield and composition of lactating Friesian × Bunaji cows supplemented with dietary vitamin E and selenium. Niger J Anim Prod. 2022;49(1):17-24.
3. Alhidary IA, Shini S, Jassim RAM, Abudabos AM, Gaughan JB. Effects of selenium and vitamin E on performance, physiological response, and selenium balance in heat-stressed sheep. J Anim Sci. 2015;93(2):576-588.
4. Arthur JR, Nicol F, Mitchell JH, Beckett GJ. Selenium and iodine deficiencies and selenoprotein function. Biomed Environ Sci. 1997;10(2-3):129-135.
5. Barwary MS, Alkass JE, Bamerny AO. Effect of diet supplemented with ascorbic acid on some physiological, haematological and biochemical constituents of blood of meriz goat. J Duhok Univ. 2011;14(1):16-24.
6. Behera HK, Jena GR, Kumar D, Mishra S, Das D, Samal L, et al. Ameliorative effect of vitamin C on hemato-biochemical and oxidative parameters in ducks during summer. Int J Livest Res. 2020;10(4):140-148.
7. Bhardwaj H, Singh C, Nayyar S, Sodhi S, Jindal R. Effect of vitamin E and selenium (Se) supplementation on biochemical parameters and expression of metallothionein (MT-2) in heavy metals exposed buffaloes. Indian J Anim Res. 2022;56(3):281-289.
8. Borges SA, Fischer AV, Silva DA, Maiorka A. Acid base balance in broilers. World Poult Sci J. 2007;63(1):73-81.
9. Department of Animal Husbandry and Dairying (DAHD). 21st livestock census. New Delhi: Government of India; 2024.
10. Duncan DB. Multiple range and multiple F tests. Biometrics. 1955;11(1):1-41.
11. Fesseha H, Ayele A. Recurrent prepartum cervicovaginal prolapse in a crossbred Holstein Friesian dairy cow. Open J Vet Med. 2020;4(3):107-109.
12. Gade NE, Singh G, Sonawane PR, Mahapatra RK. Effect of ascorbic acid supplementation on plasma protein profile in buffaloes during heat stress. Indian J Vet Res. 2010;19(2):56-62.
13. Gutteridge JM, Halliwell B. Oxidative stress, redox stress or redox success. Biochem Biophys Res Commun. 2018;502(2):183-186.
14. Helal TS, Ali FA, Ezzo O, El-Ashry MA. Effect of supplementing some vitamins and selenium during the last stage of pregnancy on some reproductive aspects of Egyptian dairy buffaloes. J Agric Sci Mansouria Univ. 2009;19(12):4289-4299.
15. Joshi KR, Pathan MM, Madhira SP, Pande AM, Dhusa DD. Study of haematological parameters of crossbred cows during peripartum period. Int J Curr Microbiol App Sci. 2018;7(12):461-467.
16. Juneja R, Jhamb D, Gaur M, Sharma SK, Sain A. A retrospective study on causes of dystocia in cattle and buffaloes at referral centre in south Rajasthan. Indian J Vet Sci Biotechnol. 2023;19(2):104-106.
17. Kannan G, Terrill TH, Kouakou B, Gelaye S, Amoah EA. Simulated preslaughter holding and isolation effects on stress responses and liveweight shrinkage in meat goats. J Anim Sci. 2002;80(7):1771-1780.
18. Kassab AY, Mohammed AA. Ascorbic acid administration as anti-stress before transportation of sheep. Egypt J Anim Prod. 2014;51(1):19-25.
19. Khan HM, Mohanty TK, Bhakat M, Gupta AK, Tyagi AK, Mondal G. Effect of vitamin E and mineral supplementation on biochemical profile and reproductive performance of buffaloes. Buffalo Bull. 2015;34(1):63-72.
20. Mahmoud GB, Abdel-Raheem SM, Hussein HA. Reproductive and physiological traits of Ossimi rams as affected by vitamin E and selenium injection. Egypt J Anim Prod. 2014;51(2):99-105.
21. Mogal IR, Razzaque WAA, Ramteke SS, Gaikwad NZ, Ali SS, Jadhav RK, et al. Effect of vitamin E and selenium on haemato-biochemical constituents in dystocia affected Marathwadi buffaloes. Int J Livest Res. 2019;9(4):146-153.
22. Norazlina M, Chua CW, Ima-Nirwana S. Vitamin E deficiency reduced lumbar bone calcium content in female rats. Med J Malaysia. 2004;59(5):623-630.
23. Pehlivan FE. Vitamin C: An antioxidant agent. In: Vitamin C. London: IntechOpen; 2017. p. 23-35.
24. Rosser B, Gores SA. Cellular mechanisms and clinical implications. Gastroenterology. 1995;108(1):252-259.
25. Semsirmboon S. The effect of high dose ascorbic acid and high DCAD administration on oxidative stress and mammary gland function of dairy goat fed under tropical condition [dissertation]. Bangkok: Chulalongkorn University; 2022. International Journal of Advanced Biochemistry Research https://www.biochemjournal.com
26. Shinde PL, Dass RS, Garg AK. Effect of vitamin E and selenium supplementation on haematology, blood chemistry and thyroid hormones in male buffalo (Bubalus bubalis) calves. J Anim Feed Sci. 2009;18(2):241-256.
27. Snedecor GW, Cochran WG. Statistical methods. 8th ed. Kolkata: Oxford and IBH Publishing Company; 2004.
28. Sultana JR, Chandra AS, Ramana DBV, Raghunandan T, Prakash MG, Venkateswarlu M. Effect of supplemental chromium, vitamin E and selenium on biochemical and physiological parameters of Holstein Friesian calves under heat stress. Indian J Anim Res. 2022;56(8):921-927.
29. Sushma K, Raman Reddy Y, Nalini Kumari N, Baswa Reddy P, Raghunandan T, Sridhar K. Effect of selenium supplementation on performance, cost economics, and biochemical profile of Nellore ram lambs. Vet World. 2015;8(10):1150-1155.
30. Tripathi A, Mehta JS. Studies on the types and prognostic approaches for uterine torsion among cattle. J Anim Res. 2016;6(1):129-134.
31. Volker L, Weiser H. The relevance of vitamins D3 and C for bone metabolism in poultry. In: Proceedings of the Maryland Nutrition Conference; 1993 Mar 25-26;
32. Weldeyohanes G, Fesseha H. Dystocia in domestic animals and its management. Int J Pharm Biomed Res. 2020;7(2):1-11.
33. White A, Estrada M, Walker K, Wisnia P, Filgueira G, Valdes F, et al. Role of exercise and ascorbate on plasma antioxidant capacity in thoroughbred race horses. Comp Biochem Physiol A Mol Integr Physiol. 2001;128(1):99-104.
34. Xiao J, Khan MZ, Ma Y, Alugongo GM, Ma J, Chen T. The antioxidant properties of selenium and vitamin E; their role in periparturient dairy cattle health regulation. Antioxidants. 2021;10(10):1555.
35. Yanardag R, Ozsoy-Sacan O, Ozdil S, Bolkent S. Combined effects of vitamin C, vitamin E, and sodium selenate supplementation on absolute ethanol-induced injury in various organs of rats. Int J Toxicol. 2007;26(6):513-523.
36. Yenilmez K, Atalay H, Cellat M, Etyemez M, Atmaca G, Gürsel FE. The effect of vitamin E administration to dairy cows in the prepartum period on some metabolic, oxidative and reproductive parameters. Large Anim Rev. 2022;28(5):235-240.
37. Youssef MM, Abdel-Aziz NA, El-Malky OM, Abd ElSalaam AM. Growth rate, metabolic activity and reproductive performance of buffaloes bulls administered with different levels of vitamin C. In: Proceeding of the 4th Scientific Conference of Animal Production Research Institute; 2013; Dokki, Giza, Egypt.