diurnal changes in blood metabolites and their relation to plasma growth hormone and time of feeding...

DIURNAL CHANGES IN BLOOD METABOLITES AND THEIR

RELATION TO PLASMA GROWTH HORMONE AND TIME

OF FEEDING IN MITHUN HEIFERS (BOS FRONTALIS)

Mohan Mondal,1 Chandan Rajkhowa,1 and B. S. Prakash2

1Animal Endocrinology Laboratory, National Research Centre on Mithun (ICAR),Jharnapani, Medziphema, Nagaland, India2Dairy Cattle Physiology Division, National Dairy Research Institute, Haryana, India

The aim of the present study was to investigate what, if any, diurnal changes occur inblood metabolites in relation to plasma growth hormone (GH) and feeding timeamong mithun (Bos frontalis), a semi-wild ruminant. Blood samples were collected athourly intervals during a 24h span from 6 mithun heifers (averaging 2.5 yr of ageand averaging 230kg in weight) that were fed twice a day at 11:00 and 16:00h.Samples were assayed for plasma GH and blood metabolites, non-esterified fattyacids (NEFA), glucose, and alpha-amino nitrogen. The total sampling period wasdivided into a 1) postprandial (after meal) period (period I: 11:00 to 21:00h) and 2)interprandial period (period II: 22:00 to 10:00h) and also into night (20:00 to05:00h) and day (06:00 to 10:00h) periods for statistical analysis. Plasma glucoseand alpha-amino nitrogen levels increased (p , 0.01), and plasma NEFA and GHdecreased (p , 0.01) after each meal. No diurnal rhythmicity was detected inplasma glucose or alpha-amino nitrogen levels. Interestingly, plasma NEFA and GHlevels were higher (p , 0.01) during the interprandial (period II) and night periods,indicating an energy deficit that occurred progressively during the interprandialperiod of nocturnal feed deprivation. In twice-daily-fed mithuns we conclude that:1) plasma metabolites and GH exhibited a definite pattern of change with time offeeding; 2) concentrations of plasma NEFA were higher nocturnally due to anenergy deficit and that GH levels were higher during the interprandial period afterthe second meal; 3) the interprandial period after the second feeding may be con-sidered to constitute a short-term food deprivation; 4) the longer interprandialperiod of 19h in this study between the second and subsequent morning meal maybe changed into equally divided feedings to minimize the short-term energy deficit;and 5) blood sampling for blood metabolites in mithuns should be conducted at afixed time of day with special emphasis on time of feeding.

Keywords Metabolites, Mithun, Growth Hormone, Feeding, Diurnal Rhythm,Ruminant

Submitted February 17, 2005, Returned for revision April 28, 2005, Accepted June 27, 2005Address correspondence to Mohan Mondal, Scientist (Animal Physiology), National Research

Centre on Mithun (ICAR), Jharnapani, Medziphema, Via Dimapur, Nagaland 797 106, India.E-mail: [email protected]

Chronobiology International, 22(5): 807–816, (2005)Copyright # 2005 Taylor & Francis, Inc.ISSN 0742-0528 print/1525-6073 onlineDOI: 10.1080/07420520500263359

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INTRODUCTION

Mithun (Bos frontalis), a species which is believed to have originatedmore than 8,000 years ago, is considered to have descended from thewild gaur (Simoons, 1984). This animal is a semi-wild ruminant species,found mainly in the North-Eastern Hills region (NEHR) of India. Thisunique livestock species is also found, although in lower numbers, inBhutan, Myanmar, Bangladesh, China, and Malaysia. This prized hillanimal of NEHR plays an important role in the economic, social, cultural,and religious life of the local tribal population under the undulating topo-graphy and adverse climatic conditions at moderately high altitude (300 to3,000m above sea level). The multifarious utility of mithun is well recog-nized. It acts as a potential source of meat, and it produces superiorquality milk. The growth rate of this animal is comparable with that ofcattle or buffalo under adequate nutrition (Pal et al., 2004). Age atpuberty varies between 22 to 30 months (26+ 4 months) and bodyweight is around 250kg. Due to the remoteness of their habitats andother ecological and socio-political factors, mithuns remain one of themost understudied ungulates.

Energy balance is one of the most critical nutritional factors impac-ting animal growth, health, lactation, and reproductive performance.Traditionally, changes in energy balance are monitored by scoringbody weight and condition. This procedure may not be sensitiveenough when dealing with growing animals (Van Saun, 2000); thus, asecond method, the measurement of beta-hydroxybutyrate (BHB) con-centration, is now favored. However, BHB concentrations may not besufficiently sensitive, and moreover, they may not be representative ofdietary sources. A third method relies of determining energy balanceinvolves the assessment of traditional parameters, such as the measure-ment of non-esterified fatty acids (NEFA). Many studies show good cor-relation between energy balance and serum NEFA concentration(Kartiarso et al., 1989; Yelich et al., 1996). Serum NEFA concentrationis the result of adipose tissue breakdown in response to negativeenergy balance; thus, it directly reflects the amount of adipose tissuebreakdown. Circulating NEFAs are absorbed and metabolized forenergy by the liver and other tissues. Clinical experience suggestsserum NEFA concentrations to be more sensitive to energy balancechanges compared to body condition scoring among growing animals(Van Saun, 2000).

Blood glucose levels have been reported to provide an index of nutri-tional status in goats (Morant-Fehr et al., 1997). Plasma glucose and NEFAare the principle circulating blood metabolites that are used to assessenergy status. Blood alpha amino nitrogen, on the other hand, is an indi-cator of protein synthesis status of the animal. Plasma alpha amino

M. Mondal, C. Rajkhowa, and B. S. Prakash808

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nitrogen has been reported to increase during growth (Hornick et al.,1996, 1998).

In cattle, metabolite and hormone concentrations generally follow acircadian rhythm, especially related to feed intake (Van Eenaeme et al.,1990; Clement et al., 1991). Diurnal patterns of metabolites and hormoneswere found to be affected by time of feeding, with an energy deficit occur-ring during the nocturnal interprandial period but not during the diurnalinterprandial period in cattle (Ndibualonji et al., 1997). Diurnal variationof some of the metabolites, such as non-esterified fatty acids (NEFA),glucose, and amino acids, has also been reported in cows (Eicher et al.,1999; Thomson et al., 2003). Diurnal changes of blood metabolites andeffect of feeding on the levels of blood metabolites have also been recordedin equines (DePew et al., 1994).

No information on diurnal rhythmicity for blood metabolites and theirrelation to plasma GH and time of feeding, if any, is available in mithuns.The present study was, therefore, framed to record the diurnal changesof blood metabolites and to establish the relationship, if any, with endo-genous plasma GH and time of feeding in mithun heifers.

MATERIALS AND METHODS

Animal Care and Management

Six growingmithun heifers averaging 2.5 yr in age (range, 2.0 to 2.8 yr)and averaging 230kg in weight (range, 212 to 253 kg) were used in thisstudy conducted on June 2 and June 4, 2003. The Mithuns were main-tained in semi-intensive condition. They were fed a mixture of locally avai-lable green grasses and tree leaves at 11:00 and 16:00h (Table 1), and theywere also fed a concentrate mixture (2.0 kg/d) consisting of crushed maizegrain (30%), wheat bran (15%), rice polish (20%), mustard cake (33%),mineral mixture (1%), and salt (1%). The chemical composition of the con-centrate mixture was organic matter (92.7%), ash (7.3%), crude protein(19.0%), ether extract (6.7%), crude fibre (4.9%), nitrogen-free extract(62.1%), total carbohydrate (69.9%), and acid detergent fibre (9.6%). All the

TABLE 1 Composition (on Dry Matter Basis in Percentage) of Green Grasses and/or Tree Leaves Fedto the Mithun Calves

Feed stuffsDry

matterOrganicmatter Ash

Etherextract

Crudeprotein

Acid detergentfiber

Albizzia sp. (21%) 44.5 95.3 4.7 2.4 15.2 31.9Borrena hirticulata (9%) 12.1 81.6 18.4 0.6 17.8 18.1Thysaenolaena maxima (13%) 27.0 94.7 8.3 1.9 12.3 37.9Curculigo recusvata (27%) 14.7 84.5 15.5 3.0 19.9 34.2Ficus hirta (20%) 26.7 83.4 16.6 1.4 14.3 32.0

Diurnal Changes in Blood Metabolites 809

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animals were born at National Research Centre on the Mithun Farmlocated in the Medziphema area of Nagaland State in India.

Blood Sampling

Blood samples (4mL) were collected bymeans of an indwelling jugularcatheter, starting at 06:00h each day and thereafter at 1 h intervals for24h, in heparinized tubes (20 IU heparin/mL of blood). Blood sampleswere centrifuged immediately and plasma stored at 2208C until assayedfor GH, non-esterified fatty acids, glucose, and alpha amino nitrogen. Allexperimental protocols and animal care met the regulations of Insti-tutional Animal Care and Use Committee (IACUC) and the standards ofthe Journal (Touitou et al., 2004). Before catheterization, local anesthesia(Xylocainew) was given, and after removal of catheter antibiotic treatment(Oxytetracyclinew) was provided for 7 days to avert infection.

Growth Hormone Assay

GH was estimated by an enzyme immunoassay developed in our labo-ratory using a second antibody technique as detailed elsewhere (Mondalet al., 2004). All assays were conducted using 25mL of mithun plasma.GH standards (USDA-bGH-B-1), prepared in charcoal treated plasmaranging from 25pg/25mL/well to 12,800pg/25mL/well, were run. Thesensitivity of the assay was 1.0 ng/mL plasma. The 50% relative binding(B/B0) sensitivity was 36.0 ng GH/mL plasma. Intra- and inter-assay coef-ficients of variation were determined using pooled plasma containing 2.0and 256.0 ng/mL and found to be 9.6, 6.8% and 8.8, 7.1%, respectively.

Estimation of Blood Metabolites

Plasma NEFA were estimated by the copper soap extraction methodmodified by Shipe and colleagues (1980) for milk. The estimation ofplasma alpha amino nitrogen was accomplished by the procedure ofGoodwin (1970) and plasma glucose was measured by the glucoseoxidase/peroxidase method using commercial kits. (Span DiagnosticsLtd., India; Product code #25940).

Statistical Analysis

The results are expressed as means and standard errors of the mean(SEM). The total sampling period was divided into two periods, i.e., a post-prandial period (after meal), from 11:00 to 21:00h (period I), and an inter-prandial period, from 22:00 to 10:00h (period II). To assess the diurnalrhythmicity of blood metabolites, data were also analyzed for two


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additional periods, during the night (22:00 to 05:00h) and daytime (06:00to 10:00h). The data from period I and period II and of the night anddaytime hours were separately analyzed by analysis of variance(ANOVA) for changes in blood metabolites with sampling time and timeof feeding. Hormone concentrations and their temporal patterns weretested for significant linear trends. Variance homogeneity was alsochecked (Snedecor and Cochran, 1980). All results were consideredsignificant at the p , 0.05 level.

RESULTS

The plasma GH and blood metabolites (NEFA, glucose, and alphaamino nitrogen) are presented in Figure 1 for individual mithuns. Thepostprandial decrease of plasma NEFA concentration was evident foreach of the mithuns. Plasma glucose and alpha amino nitrogen concen-tration was increased postprandially after each feeding in every animal.No diurnal rhythmicity was observed in plasma glucose or alpha aminonitrogen. Interestingly, plasma NEFA levels were found to be higherduring the night hours (Figures 1 and 2). Immediately after feeding, theplasma NEFA concentrations started decreasing, reaching the lowestlevels of 201.3 and 177.8mmol/L 3h after the first and second meal,respectively. Unlike plasma NEFA, plasma glucose and alpha amino nitro-gen concentration increased (p , 0.01) after both the first and secondmeal (Figures 1 and 2). An immediate postprandial increase in plasmaglucose, to a peak level of 76 and 75mg/dL, was observed 3h and 2h,respectively, after first and second feeding before decreasing thereafterto a basal value (�40mg/dL) 5h postprandially (Figure 2). Similarly,plasma alpha amino nitrogen exhibited peak values of 83 and 86mg/L3h and 2h, respectively, after first and second feeding before decreasingthereafter to a basal level (50mg/L) 5h postprandially, with minor fluctu-ation thereafter (Figure 2). Plasma GH and NEFA concentrations were sig-nificantly higher (p,0.01) during period II (interprandial) than duringperiod I (postprandial period), as shown in Table 2. Unlike plasma GHand NEFA, the plasma glucose and alpha amino nitrogen levels werefound to be significantly lower (p , 0.01) in period II than in periodI. Plasma GH was found to decrease after both meals and to increaseduring period II (Figure 2).

DISCUSSION

The results of the present study showed that the plasma glucose andalpha amino nitrogen concentrations increased and that plasma NEFAlevels decreased after the consumption of each the two daily meals. Theglucose and NEFA concentration responses were similar to those reported


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for cows (Eicher et al., 1999; Thomson et al., 2003; Nielson et al., 2003)and horses (DePew et al., 1994). The mechanism for some, but not all, ofthese responses is well documented. The elevation of the plasma glucoseconcentration following the consumption of a meal, for instance, isknown to be mainly due to the breakdown of carbohydrates supplied bythe feed resources (Guyton, 1986). Rising plasma glucose concentrationtriggers the release of insulin (DePew et al., 1994), which in turn increasesglucose uptake by tissues and reduces hepatic glucose output, therebyreturning glucose concentrations to normal physiological limits. The prob-able increase in insulin concentrations not only stimulates glucose uptake

FIGURE 1 Blood metabolites of NEFA, glucose, alpha-amino nitrogen, and GH profiles in individualsix mithun heifers. Blood samples were collected at hourly interval for 24h from 06:00 to 06:00h. Ani-mals were fed twice a day at 11:00 and 16:00h. Sunrise was at 05:12h and sunset at 17:40h. The arrowsdesignate time of feeding.


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but also inhibits the release of fatty acids from adipose tissues by decreasingthe activity of hormone-sensitive lipase (Brockman and Laarveld, 1986),resulting in a rapid short-term decrease in plasma NEFA concentrations,as observed in the present study after each meal.

In the present investigation, the plasma alpha amino nitrogen concen-trations were found to increase after each meal. Given that no diurnal varia-tion of alpha amino nitrogen was observed in the mithuns, the source ofthe elevated postprandial concentrations of alpha amino nitrogen wasfeeding. Alpha amino nitrogen is reported to be absorbed across theruminal tissues (30% of total net absorption) and intestinal tissues (70%of the total net absorption) in cattle (Theurer et al., 2002).

Plasma GH decreased postprandially, and this finding is consistentwith those from cattle (Hove and Bloom, 1973; Ndibualonji et al., 1997)

FIGURE 2 Blood metabolites of NEFA, glucose, alpha-amino nitrogen, and GH levels shown inFigure 1 were averaged and presented in this Figure with standard errors. The arrows designatetime of feeding.

TABLE 2 Mean (+SEM) Plasma GH (ng/mL), NEFA (mmol/L), Glucose (mg%), and Alpha AminoNitrogen (mg/L) During Postprandial (Period I) and Interprandial Period (Period II)

Period GH NEFA GlucoseAlpha aminonitrogen

Period I 64.0+ 6.9a 229.3+ 8.2a 58.6+ 5.9a 69.8+ 6.2a

Period II 74.4+ 9.4a 310.2+ 10.6a 39.9+ 4.6a 53.6+ 5.7a

Overall 69.9+ 10.1 274.6+ 14.8 48.1+ 6.6 60.8+ 7.3

aMeans for Period I and Period II differed significantly (p , 0.01).


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and sheep (Trenkle, 1971a, 1971b, 1978; Bassett, 1972). In those species,GH concentrations decreased after feeding and remained low for 1 to 2hthereafter. In contrast, feed deprivation producedmore frequent episodesof GH release (Hove and Bloom, 1973; Bassett, 1974). In one study con-ducted on sheep, a 60% reduction in GH concentrations was observedwithin 45min after the meal began (Trenkle, 1989). The plasma GHincrease over time after 21:00h (following feeding at 16:00h) and the sim-ultaneous overnight increase in plasma NEFA confirm an energy deficitduring the nocturnal interprandial period, which may, therefore, be con-sidered as short-term effect of feed deprivation. The elevated nocturnalNEFA concentrations were due to an energy deficit of feed deprivationresulting in increased lipolysis from adipose tissue, which is indicatedalso by higher plasma GH levels during the corresponding time span.These observations clearly suggest that the time from last meal, whichwas about 19h apart for this study, rather than photoperiod, influencesthe patterns of NEFA and GH concentration in mithuns. There is nodoubt that in twice-daily-fed mithuns, an energy deficit occurs duringthe interprandial period, and that the animals respond by increasingplasma GH. Still, normoglycemia was maintained during the interprandialperiod despite an energy deficit, like as a result of increased GH, whichfavors gluconeogenesis from amino acids (proteolysis) and glycerol(lipolysis; Ndibualonji and Godeau, 1993).

In conclusion, the results reported in the present study indicate that 1)plasma metabolites and GH exhibit definite pattern of change with time offeeding; 2) nocturnally observed higher concentrations of plasma NEFAwere due to an energy deficit and higher GH levels during interprandialperiod after the second meal; 3) the interprandial period after thesecond feeding may be considered as a short-term feed deprivation;4) the longer interprandial period between the second meal and nextmorning meal, which was 19h apart in this study, may be equallydivided throughout the day to minimize the short-term energy deficit;and 5) blood sampling for blood metabolites in mithuns should be con-ducted at a fixed time of the day with special emphasis on time of feeding.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Mark Hennies, Institute of Tier-anatomic, Bonn, Germany for the generous gift of highly specific bGHantibody. The supply of highly purified reference preparation of bovineGH by USDA Animal Hormone Program, Beltsville, Maryland, USA isgratefully acknowledged. The authors also wish to thank the Director,National Dairy Research Institute, Karnal-132 001 (Haryana), India forproviding a part of facilities for the present work.


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diurnal changes in blood metabolites and their relation to plasma growth hormone and time of feeding...

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