Effect of dietary energy levels on performance and blood biochemical parameters of deccani ewes during summer

Introduction

Sheep play an important role in the livelihood of rural communities in India by providing meat, wool, skin and manure. Among the indigenous breeds, Deccani sheep are well adapted to the semi-arid regions of southern India and contribute significantly to mutton production. However, high ambient temperatures during summer may adversely affect feed intake, nutrient utilization and animal performance. Energy is the most critical nutrient influencing maintenance and productive functions in sheep. Under heat stress conditions, changes in energy metabolism may alter nutrient requirements and affect animal performance. Information on the optimum dietary energy requirement of Deccani sheep during summer is limited. Therefore, the present study was undertaken to evaluate the effect of varying dietary energy levels on feed intake and body weight changes of Deccani ewes during summer conditions.

Ewes were randomly allocated into three treatment groups of eight animals each under a completely randomized design. Animals were housed in a well-ventilated shed with facilities for individual feeding and watering. Prior to the commencement of the experiment, all animals were dewormed and vaccinated against sheep pox, peste des petits ruminants and enterotoxaemia as per standard schedules. Three sorghum stover-based complete diets were formulated: 1. NE: Maintenance energy level (ICAR, 2013) [1] , 2. HE-10: 10% higher energy than maintenance, 3. HE-20: 20% higher energy than maintenance (Table 1). All diets were iso-nitrogenous and met nutrient requirements except for energy concentration. During the first 60 days, animals received their respective experimental diets at 3.1% of body weight. From day 61 to the 108th day of gestation, all ewes were fed the maintenance diet at 3.1% of body weight. During the final six weeks of gestation until lambing, the maintenance diet was offered at 3.73% of body weight to meet increased nutrient requirements.

Proximate Analysis

The proximate analysis of feeds (Organic Matter, Crude Protein, Ether Extract, Crude Fibre, Nitrogen Free Extract and Total Ash Content (%)) was performed as per the procedures described in AOAC (2012) [2] .

Feeding and data collection

Feed was offered twice daily at 10:00 AM and 2:30 PM in equal proportions. Fresh drinking water was provided ad libitum. Feed offered and refusals were recorded daily for estimation of feed intake and dry matter intake. Body weights were recorded at fortnightly intervals before feeding and watering.

Blood biochemical parameters

Blood was collected from individual ewes on 45th and 120th day of experiment and 15th day before expected day of lambing. Blood samples were collected aseptically from jugular vein of ewes into clean sterilized glass tubes and kept in slanted position at room temperature for separation of serum. The collected serum samples were centrifuged at 3000 rpm for 5 minutes and transferred to 5 ml Eppendorf tubes and stored at -20oC for estimation of various biochemical constituents. Serum biochemical parameters were estimated by using a commercial kit (Coral Clinical Systems, Goa) and following methods: 1) Serum glucose by enzymatic method (Trinder, 1969a) [3,4] , 2) Serum total protein by Biuret method (Reinhold, 1953) [5] , 3) Serum albumin by Bromocresol green (BCG) method (Doumas et al., 1971) [6] , 4) Serum globulin was calculated by using formula Globulin (g/dl) = Total proteins (g/dl) -Albumin (g/dl), 5) Albumin-Globulin ratio was calculated by following formula: Albumin -globulin ratio (A/G) ratio = Albumin (g/dl) / Globulin (g/dl), 6) Serum cholesterol was estimated using commercial kit. Values obtained were calculated by (Absorbance of Test/Absorbance of Standard) x Conc. of Standard (mg/dl) (Allain et al., 1974) [7] , 7) Serum triglycerides by Glycerol phosphate oxidase (GPO) method (Trinder, 1969b) [3,4] and 8) Blood urea nitrogen was estimated by enzymatic method (Bretaudiere et al., 1976) [8] using kit supplied by Accurex Biomedical Private Limited, Boisar, Maharastra

Statistical analysis

Data were analyzed using SPSS Version 15.0. One-way and two-way analysis of variance under the generalized linear model procedure was employed. Treatment means were compared using Duncan's Multiple Range Test (Duncan, 1955) [9] and significance was declared at P<0.05. All the statistical procedures were done as per Snedecor and Cochran (1994) [10] .

Results and discussion 1. Chemical composition of diets

The chemical composition of the sorghum stover-based complete diets is as shown in Table 2. The organic matter, crude protein, ether extract, crude fibre, nitrogen free extract and total ash content (%) of NE diet on dry matter basis was 89.96, 9.72, 1.64, 30.17, 48.43 and 10.04 respectively. The corresponding values for HE-10 diet was 91. 30 2013) [1] . HE10-10% higher energy than the maintenance requirement. HE20-20% higher energy than the maintenance requirement.

Feed intake and dry matter intake

Dietary energy levels did not significantly influence feed intake or dry matter intake during the first 60 days of the experiment (Table 3 and4). Average feed intake during the first fortnight was 819.8, 818.9 and 818.9 g/day for NE, HE-10 and HE-20 groups, respectively. Corresponding dry matter intakes were 737.8, 737.0 and 737.0 g/day. Similar trends were observed throughout the first four fortnights. Following the treatment period, all animals received the maintenance diet and feed as well as dry matter intakes remained comparable among groups until lambing. The absence of differences in feed intake among dietary treatments suggests that increasing dietary energy concentration by 10 or 20% above maintenance did not alter voluntary feed consumption in Deccani ewes. During the study period, Temperature Humidity Index (THI) values ranged from 89.76 to 94.38, indicating severe heat stress conditions. Heat stress is generally associated with reduced feed intake as animals attempt to minimize metabolic heat production. However, Deccani sheep maintained normal intake, likely due to their adaptation to hot climates, provision of adequate shade and unrestricted access to drinking water. These findings agree with reports of Giger-Reverdin et al. (2013) [11] and Fiaz et al. (2012) [12] , who observed no significant effect of dietary energy level on feed intake under heat stress conditions. Similar observations have also been reported by Rekik et al. (2008) [13] and Hossain et al. (2003) [14] . [1] ; HE 10: High energy diet 1; HE 20: High energy diet 2 during first 60 days of feeding period. SEM: Standard Error Mean; P value: Probability value; N: Number of observations. [1] ; HE 10: High energy diet 1; HE 20: High energy diet 2 during first 60 days of feeding period. SEM: Standard Error Mean; P value: Probability value; N: Number of observations.

Body Weight Changes

No significant differences were observed in the body weights among treatment groups throughout the experimental period (Table 5). The maintenance energy level recommended by ICAR (2013) [1] , equivalent to approximately 127 kcal ME/kg W^0.75, was sufficient to sustain body weight and support normal physiological functions under severe summer conditions. Increasing dietary energy by 10 or 20% above maintenance did not improve body weight gain. The gradual increase in body weight observed during the later stages of the experiment was mainly attributable to pregnancy and fetal growth rather than treatment effects. Similar findings have been reported by Macías-Cruz et al. (2016) [15] , Hossain et al. (2003) [14] and Koyuncu and Canbolat (2009) [16] , who observed no significant changes in body weight with moderate increases in dietary energy. In contrast, studies involving substantially higher energy supplementation and ad libitum feeding have reported improved body weight gains (Kaminski et [17,18,19] . [1] ; HE 10: High energy diet 1; HE 20: High energy diet 2 during first 60 days of feeding period. SEM: Standard Error Mean; P value: Probability value; N: Number of observations.

Serum biochemical parameters 4.1 Serum glucose, serum triglycerides and serum cholesterol

Variation in dietary energy levels had no effect on serum glucose on 45th and 120th day, while on 15thday before expected day of lambing, the concentration of glucose in serum was lower (P<0.01) in HE-10 and HE-20 group ewes when compared to NE group ewes. No significant effect was observed on serum triglycerides and serum cholesterol by the supplementation of different dietary energy levels (Table and6.1).

However, the time of blood collection irrespective of diets influenced blood glucose, triglycerides and serum cholesterol values. Significantly (P<0.01) highest serum glucose value was observed on 45 th day (85.09 mg/dl), followed by 15 th day before expected day of lambing (70.54 mg/dl) and lowest levels were observed on 120 th day (64.93 mg/dl) of blood collection. Serum triglyceride values were significantly (P<0.01) higher on 120 th day and 15 days before lambing and lower on 45 th day. Whereas, serum cholesterol values were significantly (P<0.01) higher on 45 th day, followed by 120 th day and lower on 15 days before lambing. The elevated glucose concentration on day 45 may be attributed to heat stress-induced glycogenolysis mediated by glucocorticoids (Thompson, 1973) [20] , whereas the decline during late gestation likely reflects increased maternal glucose utilization and fetal glucose demand (Duehlmeier et al., 2013) [21] . The absence of a consistent dietary effect on glucose agrees with previous studies reporting minimal changes following energy supplementation (Karikari and Blasu, 2009; Kawardeep et al., 2013; Hashem and El-Zarkouny, 2013) [22,23,24] , although other authors have reported increased glucose concentrations with high-energy diets (Mosaad and [25,26,27] . Serum cholesterol and triglyceride concentrations were not affected by dietary energy level, indicating that moderate increases in dietary energy did not alter lipid metabolism. However, irrespective of treatment, serum cholesterol declined progressively from day 45 to late gestation, while triglyceride concentrations increased on day 120 and 15 days before lambing. The reduction in cholesterol may reflect its utilization for steroid hormone synthesis and fetal development, while increased triglycerides are consistent with metabolic adaptations during pregnancy (Deghnouche et al., 2013; Piccione et al., 2009) [28,29] . Similar findings have been reported by Kaminski et al. (2015) [17] , whereas increased lipid concentrations with higher energy diets have also been documented under different nutritional regimens like high energy diets (Senosy et al., 2013; Habibizad et al., 2015) [27,30] , high protein diets (Mahmoud et al., 2014) [31] or supplemented with fat (Hashem and El-Zarkouny, 2013) [24] or by flushing (Kia and Safdar, 2015 and Kia et al., 2016) [32, 33] . [1] ; HE 10: High energy diet 1; HE 20: High energy diet 2 during first 60 days of feeding period. 45d and 120d: Blood collected on 45th and 120th day of experiment; 15d BL: 15th day from expected day of lambing. SEM: Standard Error Mean; P value: Probability value; N: Number of observations.

Serum protein, albumin, globulin, albumin-globulin ratio and blood urea nitrogen

Variation in dietary energy levels had significant (P<0.01) effect on serum total protein where higher dietary energy levels (HE-10 and HE-20) showed lower serum protein values when compared to control during 15 days before lambing (Table 7 and7.1). Whereas no effect was observed during 45 th day and 120 th day. Supplementation of NE, HE-10 and HE-20 diets showed no significant effect on the serum albumin concentration at 45 th , 120 th day and 15 days before lambing. The serum globulin content did not differ in ewes of NE, HE-10 and HE-20 groups on 45th and 120th day of feeding trial, while at 15 days before expected day of lambing, the globulin content was significantly (P< 0.05) higher in ewes of NE group than ewes of HE-10 and HE-20 groups. The serum albumin-globulin ratio values did not differ significantly on 45 th day of feeding trail. However, on 120 th day of trial, the serum albumin-globulin ratio was significantly (P< 0.05) lower in ewes of HE-10 group and higher in ewes of HE-20 group, while the ratio was intermediate in ewes of NE group. The serum albuminglobulin ratio in ewes at 15 days before expected day of lambing was significantly lower (P< 0.05) in NE and HE-10 groups and higher in ewes of HE-20 group. Variation in dietary energy had no effect on serum blood urea nitrogen concentration on 45 and 120 th day of trial and at 15 days before expected day of lambing. Irrespective of the diets supplemented to the ewes, all the serum biochemical paramters showed significant (P<0.05) effect during 45 th day, 120 th day and 15 days before lambing of trial (Table 6). The serum total protein and serum globulin levels were significantly (P< 0.01) lower on 120 th day of feeding trial and 15 th day before lambing and higher on 45 th day of feeding. The albumin and blood urea nitrogen levels significantly (P< 0.01) lower on 45 th day and 15 th day before expected day of lambing and were higher on 120 th day of collection. The albumin-globulin ratio was significantly (P< 0.01) higher during 3rd collection (15 days before expected day of lambing) and lower on day 120 of feeding trial with intermediate values observed on 45th day of trial. Dietary energy had no effect on serum total protein, albumin, globulin, or albumin:globulin (A:G) ratio during early and mid-pregnancy. However, total protein and globulin concentrations were lower in the high-energy groups during late gestation, while albumin and the A:G ratio remained unaffected, suggesting maintenance of protein homeostasis despite changes in globulin levels. This reduction may be associated with increased fetal protein demand and greater nutrient partitioning towards conceptus growth during advanced pregnancy (Lloyd et al., 2012; Duehlmeier et al., 2013) [34,21] . Irrespective of dietary treatment, serum protein and globulin concentrations declined as gestation advanced, whereas albumin and the A:G ratio remained relatively stable, indicating that physiological stage exerted a greater influence than dietary energy level. Similar findings of unchanged albumin and total protein following energy supplementation have been reported in ewes by Mosaad and Derar (2009) [25] , Naqvi et al. (2011) [35] and Kia and Safdar (2015) [32] and in goats by Kawardeep et al. (2013) [23] . Likewise, Piccione et al. (2009) [29] and Antunović et al. (2002) [36] reported that serum albumin remains relatively stable during gestation, whereas globulin concentrations may decline because of increased fetal nutrient demand and physiological adaptations. In contrast, Senosy et al. (2013) [27] and Khalifa et al. (2015) [18] observed increased serum protein and globulin concentrations in ewes receiving high-energy diets, suggesting that the response may depend on the level and duration of nutritional supplementation. Blood urea nitrogen was unaffected by dietary energy level but varied with stage of gestation, with the highest concentrations observed on day 120. The absence of dietary effects indicates that moderate energy supplementation did not markedly alter protein metabolism, while temporal changes likely reflect physiological adaptations during pregnancy. Similar observations of unchanged blood urea concentration with dietary energy supplementation have been reported by Habibizad et al. (2015) [30] , Senosy et al. (2013) [27] , and Hashem and El-Zarkouny (2013) [24] . Likewise, Nazifi et al. (2003) [37] and Ghosh et al. (2013) [38] reported minimal effects of environmental stress on blood urea concentration in sheep and goats, respectively. Conversely, Antunovic et al . ( 2002) [36] , Indu et al. (2014) [39] , and Rathwa et al. (2017) [40] observed increased blood urea concentrations during heat stress. Overall, the present findings suggest that physiological stage rather than moderate dietary energy supplementation had a greater influence on protein metabolism during gestation. [1] ; HE 10: High energy diet 1; HE 20: High energy diet 2 during first 60 days of feeding period. 45d and 120d: Blood collected on 45th and 120th day of experiment; 15d BL: 15th day from expected day of lambing. SEM: Standard Error Mean; P value: Probability value; N: Number of observations.

Conclusion

The present study demonstrated that Deccani sheep maintained normal performance despite exposure to severe heat stress conditions. Feeding ewes according to the maintenance energy requirement recommended by ICAR (2013) [1] , equivalent to approximately 127 kcal ME/kg W^0.75, was adequate for performance and adaptation during summer. Therefore, additional energy supplementation above maintenance is not economically justified under similar management conditions.

Acknowledgement

AcknowledgmentsThe authors are thankful to the Department of Animal Nutrition, Department of Veterinary Gynaecology,

References

1. ICAR. Nutrient requirements of sheep, goat and rabbit. New Delhi: Indian Council of Agricultural Research; 2013.
2. AOAC. Official methods of analysis of AOAC International. 19th ed. Gaithersburg, MD: AOAC International; 2012.
3. Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem. 1969;6(1):24-27.
4. Trinder P. Enzymatic colorimetric determination of triglycerides by GOP-PAP method. Ann Clin Biochem. 1969;6:24-27.
5. Reinhold J. Determination of serum total protein, albumin and globulin fractions by the biuret method. In: Practical Clinical Biochemistry. Vol. 1. 1953:545-547.
6. Doumas BT, Watson WA, Biggs HG. Albumin standards and the measurement of serum albumin with bromcresol green. Clin Chim Acta. 1971;31(1):87-96.
7. Allain CC, Poon LS, Chan CS, Richmond WFPC, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20(4):470-475.
8. Bretaudiere JP, Phung HT, Bailly M. Direct enzymatic determination of urea in plasma and urine with a centrifugal analyzer. Clin Chem. 1976;22(10):16141617.
9. Duncan DB. Multiple range and multiple F tests. Biometrics. 1955;11(1):1-42.
10. Snedecor GW, Cochran WG. Statistical methods. 8th ed. New Delhi: Oxford & IBH Publishing; 1994.
11. Giger-Reverdin S, Jaber L, Tessier JA, Duvaux-Ponter C. Effect of a heat stress episode on feed and water intake in dairy goats bred under temperate climate. In: Proceedings of the 14th International Seminar of the FAO-CIHEAM Sub-Network on Sheep and Goat Nutrition: Feeding and management strategies to improve livestock productivity, welfare and product quality under climate change; 2013. p.107.
12. Fiaz M, Abdullah M, Pasha TN, Jabbar MA, Babar ME, Bhatti JA, et al. Evaluating varying dietary energy levels for optimum growth and early puberty in Sahiwal heifers. Pak J Zool. 2012;44:625-634.
13. Rekik M, Lassoued N, Salem HB, Mahouachi M. Effects of incorporating wasted dates in the diet on reproductive traits and digestion of prolific D'Man ewes. Anim Feed Sci Technol. 2008;147(1-3):193-205.
14. Hossain ME, Shahjalal M, Khan MJ, Hasanat MS. Effect of dietary energy supplementation on feed intake, growth and reproductive performance of goats under grazing condition. Pak J Nutr. 2003;2(3):159-163.
15. Macías-Cruz U, Gastélum MA, Álvarez FD, Correa A, Díaz R, Meza-Herrera CA, et al. Effects of summer heat stress on physiological variables, ovulation and progesterone secretion in Pelibuey ewes under natural outdoor conditions in an arid region. Anim Sci J. 2016;87(3):354-360.
16. Koyuncu M, Canbolat O. Effect of different dietary energy levels on the reproductive performance of Kivircik sheep under a semi-intensive system in the South-Marmara region of Turkey. J Anim Feed Sci. 2009;18(4):620-627.
17. Kaminski SL, Redmer DA, Bass CS, Keisler DH, Carlson LS, Vonnahme KA, et al. The effects of diet and arginine treatment on serum metabolites and selected hormones during the estrous cycle in sheep. Theriogenology. 2015;83(5):808-816.
18. Khalifa EI, Hafez YH, Mahrous AA, Behery HR, Hassanien HA, Fayed AA. Impact of non-conventional energy sources in ration on productive and reproductive performance of ewes. Egypt J Sheep Goat Sci. 2015;65:1-13.
19. Naqvi SMK, Sejian V, Karim SA. Effect of feed flushing during summer season on growth, reproductive performance and blood metabolites in Malpura ewes under semiarid tropical environment. Trop Anim Health Prod. 2013;45(1):143-148.
20. Thompson GE. Climatic physiology of cattle. J Dairy Res. 1973;40(3):441-473.
21. Duehlmeier R, Fluegge I, Schwert B, Ganter M. Insulin sensitivity during late gestation in ewes affected by pregnancy toxemia and in ewes with high and low susceptibility to this disorder. J Vet Intern Med. 2013;27(2):359-366.
22. Karikari PK, Blasu EY. Influence of nutritional flushing prior to mating on the performance of West African Dwarf goats mated in the rainy season. Pak J Nutr. 2009;8(7):1068-1073.
23. Kawardeep K, Rajesh J, Shashi N. Effect of nutritional manipulation after mating on conception rate and blood biochemical profile in Beetal goats. Indian J Small Rumin. 2013;19(1):104-106.
24. Hashem NM, El-Zarkouny SZ. Effect of short-term supplementation with rumenprotected fat during the late luteal phase on reproduction and metabolism of ewes. J Anim Physiol Anim Nutr. 2013;98(1):65-71.
25. Mosaad GM, Derar DR. Effect of dietary energy and phosphorus on nutrients digestibility, blood constituents, and ovarian structures in ewes. Vet World. 2009;2(12):456-461.
26. Zabuli J, Tanaka T, Lu W, Kamomae H. Intermittent nutritional stimulus by short-term treatment of highenergy diet promotes ovarian performance together with increases in blood levels of glucose and insulin in cycling goats. Anim Reprod Sci. 2010;122(3-4):288293.
27. Senosy W, Abdel-Raheem SM, Abd-Allah M, Fahmy S, Hassan EH, Derar RI. Effect of transient high-energy diets just after ovulation on ovarian performance and metabolic status in cyclic ewes. Small Rumin Res. 2013;109(2):152-155.
28. Deghnouche K, Tlidjane M, Meziane T, Touabti A. Influence of physiological stage and parity on energy, nitrogen and mineral metabolism parameters in the Ouled Djellal sheep in the Algerian Southeast arid area. Afr J Agric Res. 2013;8(18):1920-1924.
29. Piccione G, Caola G, Giannetto C, Grasso F, Runzo SC, Zumbo A, et al. Selected biochemical serum parameters in ewes during pregnancy, post-parturition, lactation and dry period. Anim Sci Pap Rep. 2009;27(4):321-330.
30. Habibizad J, Riasi A, Kohram H, Rahmani HR. Effect of feeding greater amounts of dietary energy for a shortterm with or without eCG injection on reproductive performance, serum metabolites and hormones in ewes. Anim Reprod Sci. 2015;160:82-89.
31. Mahmoud GB, Abdel-Raheem SM, Hussein HA. Follicular dynamics, steroid hormones and blood metabolites concentrations during long-term protein flushing in subtropical ewes. Aust J Basic Appl Sci. 2014;8(18):207-216.
32. Kia HD, Safdar AHA. Effects of calcium salts of fatty acids (CSFA) with different profiles (ω3 and ω6) during the flushing period on reproductive performance of Afshari ewes. Small Rumin Res. 2015;126:1-8.
33. Kia HD, Fazel AA, Khani AH. Different sources of protein effect in the flushing rations on some blood parameters and the reproductive performance of Ghezel sheep. Iran J Appl Anim Sci. 2016;6(3):629-638.
34. Lloyd LJ, Foster T, Rhodes P, Rhind SM, Gardner DS. Protein-energy malnutrition during early gestation in sheep blunts fetal renal vascular and nephron development and compromises adult renal function. J Physiol. 2012;590(2):377-393.
35. Naqvi SMK, Soren NM, Karim SA. Effect of concentrate supplementation on performance, ovarian response, and some biochemical profile of Malpura ewes. Trop Anim Health Prod. 2011;43(5):905-913.
36. Antunović Z, Senčić Đ, Šperanda M, Liker B. Influence of the season and the reproductive status of ewes on blood parameters. Small Rumin Res. 2002;45(1):39-44.
37. Nazifi S, Saeb M, Rowghani E, Kaveh K. The influences of thermal stress on serum biochemical parameters of Iranian fat-tailed sheep and their correlation with triiodothyronine (T3), thyroxine (T4), and cortisol concentrations. Comp Clin Pathol. 2003;12(3):135-139.
38. Ghosh S, Singh AK, Haldar C. Adaptive and ecological significance of the seasonal changes in hematological, biochemical and hormonal parameters in the tropical goat (Capra hircus). J Endocrinol Reprod. 2013;17(2):113-122.
39. Indu S, Sejian V, Naqvi SMK. Impact of simulated heat stress on growth, physiological adaptability, blood metabolites and endocrine responses in Malpura ewes under semiarid tropical environment. Anim Prod Sci. 2014;55(6):766-776.
40. Rathwa SD, Vasava AA, Pathan MM, Madhira SP, Patel YG, Pande AM. Effect of season on physiological, biochemical, hormonal, and oxidative stress parameters of indigenous sheep. Vet World. 2017;10(6):650.