2006 Zoological Society of JapanZOOLOGICAL SCIENCE

23

: 633–639 (2006)

Development of a Biotin-streptavidin Amplified Enzyme Immunoassay for Oxytocin and its Application During

Milk Ejection and the Reproductive Cyclein the Mithun (

Bos frontalis

)

Mohan Mondal

1

*,

Chandan Rajkhowa

1

and Bukkaraya Samudram Prakash

2

1

Animal Endocrinology Laboratory, National Research Centre on Mithun (ICAR),Jharnapani, Medziphema, Nagaland- 797 106, INDIA

2

Dairy Cattle Physiology Division, National Dairy Research Institute,Karnal 132 001 (Haryana), INDIA

Oxytocin is a key hormone involved in milk ejection. It plays a key role in regulation of reproductivecyclicity in female mammals by taking part in the process of luteolysis. Determination of oxytocinis, therefore, important for studying the control of its secretion and its role in reproduction of themithun. A simple and sufficiently sensitive enzyme immunoassay (EIA) for oxytocin determinationin mithun plasma using the biotin-streptavidin amplification system and second antibody coatingtechnique was therefore developed. Biotin was coupled to oxytocin and used to bridge betweenstreptavidin-peroxidase and the immobilized oxytocin antiserum in a competitive assay. The EIAwas conducted directly in 200 µµµµ

l of unknown mithun plasma. Standards prepared in hormone-freeplasma were used. The lowest detection limit was 0.5 pg/ml plasma. Plasma volumes for the EIA(50, 100, and 200 µµµµ

l) did not influence the shape of standard curve, even though a drop in OD

450

was seen with higher plasma volumes. A parallelism test was carried out to compare endogenousmithun oxytocin with a bovine oxytocin standard. The former showed good parallelism with thebovine standard curve. For biological validation of the assay, plasma oxytocin was measured in theblood samples collected before, during, and after milking in three mithun cows and in six non-lac-tating cyclic mithuns during the entire estrous cycle. A sharp release of oxytocin shortly afterudder stimulation was observed. A high level of oxytocin was maintained during milking, fallingsharply thereafter. The mean plasma oxytocin concentration was different on different days of theestrous cycle (P<0.001). Two peaks of oxytocin were recorded, one at day 6 and another at day 18of the estrous cycle. In conclusion, a simple, sufficiently sensitive and direct EIA procedure hasbeen developed for the first time to determine plasma oxytocin levels in mithuns. Apart from beingnon-radioactive, the EIA procedure described here also utilizes a highly stable biotinalyted hor-mone which has a shelf life of several years, unlike the short shelf life of iodinated tracer used inRIA procedures.

Key words:

oxytocin, enzyme immunoassay, mithun, estrous cycle, milking

INTRODUCTION

The mithun (

Bos frontalis

) is believed to have originatedmore than 8000 years ago, and is considered to havedescended from wild gaur (Simoons, 1984). This is sup-ported by the resemblance, similar distribution, and absenceof a sterility barrier between the mithun and the gaur (Mon-dal and Pal, 1999). Moreover, the existence of a sterility bar-rier between mithun and cattle, different haemoglobin geno-types in mithun and zebu, and a karyotype different fromthat of European cattle, but similar to that of the gaur, also

argue in favor of the gaur origin of the mithun (Mondal andPal, 1999). The mithun belongs to Family Bovidae, Surbor-der Ruminantia (true ruminants), Order Artiodactyla, Sub-class Prototheria, Class Mammalia.

This prized hill animal is a semi-wild ruminant species,found mainly in the northeastern hilly region (NEHR) ofIndia, and in some locations in Bhutan, Myanmar, Bang-ladesh, China, and Malaysia. The mithun plays importanteconomic, social, and cultural roles in the lives of local pop-ulations, as a source of meat and milk. However, due to theremoteness of its range and other ecological and socio-polit-ical factors, the mithun remains one of the least-studiedungulates.

Oxytocin is considered to be a key hormone involved inthe process of milk ejection in lactating animals (Bruckmaierand Blum, 1998). Since the discovery of oxytocin releasefrom the ovaries and corpus luteum during the 1980s, the

* Corresponding author. Phone: +91-3862-247327 Ext 43,+91-9436004761(Mobile);

Fax : +91-3862-247341;E-mail: mohan_mondal@rediffmail.com

doi:10.2108/zsj.23.633

M. Mondal

et al

.634

hormone has also been thought to play a major role in theregulation of reproductive cyclicity in female mammals by itsinvolvement in the process of luteolysis (Silvia, 1999).Determination of oxytocin levels in peripheral circulation istherefore very important for studying the control of its secre-tion and its role in mithun reproduction. This type of informa-tion, to the best of our knowledge, is not available for anyphysiological state in this species, likely due to lack of a sim-ple assay procedure to measure the hormone. Hence, wedecided to develop and test an efficient and inexpensiveenzyme immunoassay for oxytocin in mithun plasma and toapply this procedure to determine oxytocin profiles duringmilk ejection and reproductive cyclicity in the mithun.

MATERIALS AND METHODS

Experimental animals

A total of six non-lactating mithun cows that were in regularcyclicity and three lactating mithuns weighing 254 to 351 kg, and 4to 7 years in age, were selected for the experiment from theNational Research Centre on Mithun Farm located in the Medz-iphema area, Nagaland State, India. The animals selected for thestudy were free from any anatomical and reproductive disordersand were healthy. The mithuns were maintained under semi-inten-sive conditions. They were fed

ad libitum

with mixture of locallyavailable green grasses and leaves of

Albizzia

sp.,

Borrena hirticu-lata

,

Thysaenolaena maxima

,

Curculigo recusvata

, and

Ficus hirta

trees. In addition, all mithuns were fed with a concentrate mixture(92.7% organic matter, 19.0% crude protein, 6.6% ether extract,4.9% crude fiber, 62.1% nitrogen free extract, 69.9% total carbohy-drate, and 9.6% acid-detergent fiber) in the amount of 2.0 kg perday per animal. The study was conducted from May to July 2005.The temperature and relative humidity recorded during the experi-ment were 25 to 31

°

C and 66 to 91%, respectively.

Blood sampling

Blood samples (5 ml) were collected in heparinised tubes (20IU heparin per ml of blood) by jugular venipuncture from six cyclingnon-lactating mithuns for 30 days to estimate plasma oxytocin andprogesterone levels for an entire reproductive cycle. Blood sampleswere centrifuged immediately and the plasma stored at –20

°

C untilassayed for hormones. All experimental protocols and animal carefollowed the regulations of the Institutional Animal Care and UseCommittee (IACUC).

Milking experiment

Three lactating mithuns were used in this experiment. Serialblood samples (3.5 ml) were taken from the jugular vein through anindwelling jugular catheter (a) before milking stimulus; (b) at milkingstimulus; (c) during milking; (d) after the end of milking. Blood sam-ples were taken at 1-min intervals starting from 5 min before appli-cation of the milking stimulus. During the milking stimulus and milk-ing, blood samples were collected at 30-second intervals until theend of milking, and thereafter at 1-min intervals until 5 min after theend of milking.

The samples were centrifuged immediately for 30 minutes at4

°

C. The plasma was stored at –20

°

C until analysis. Every effortwas made to minimize stress to the animals during sampling.

Analysis of the samples

To monitor cyclicity and determine the day of estrus, sampleswere analysed for progesterone by radioimmunoassay. The oxyto-cin profile was analysed by enzyme immunoassay using the secondantibody coating technique and the biotin-streptavidin amplificationsystem detailed below.

Progesterone assay

The plasma progesterone level was estimated by the simple,sensitive radioimmunoassay detailed by Mondal

et al.

(2005). Theprogesterone antiserum used in the assays was BSP NR#2(20.11.93), which was raised at the National Dairy Research Insti-tute, Karnal, India (Prakash and Madan, 2001). Twenty-microliterplasma samples were used for the assays. The minimum detectionlimit of the assay for progesterone was 2 pg/tube, which corre-sponded to 0.1 ng/ml, and the 50% binding limit was 63 pg/tube.The intra- and inter-assay coefficients of variation of plasmaprogesterone were 6.7 and 11.1%, respectively. The progesteroneantiserum (anti-progesterone-11

α

-hemisuccinate-BSA) cross-reacted with 4-pregnene-3,20 dione, 11

α

-hydroxyprogesterone,and corticosterone to the extent of 100, 110, and 0.2%, respec-tively. Cross reactivity of the antiserum with cortisone and hydrocor-tisone was <0.01%, and with

β

-estradiol, estriol, and testosterone,<0.001%.

Enzyme immunoassay of oxytocin

Preparation of affinity-purified goat IgG to rabbit IgG

Affinity-purified goat IgG to rabbit IgG was developed followingthe procedure of Anandlaxmi and Prakash (2001). Briefly, about 40ml plasma from goats immunized against rabbit IgG containing 20IU heparin/ml of blood was vortexed with rabbit IgG agarose andloaded onto a small column. Non-specific proteins were first elutedwith PBS buffer (50 mM NaPO

4

, 0.15 M NaCl, pH 7.2). Proteinsbound specifically were eluted with 15 ml of 0.1 M glycine-HCl (pH2.0). All steps were performed at room temperature. The elutedfractions (3 ml each) were collected in vials containing 0.2 ml of 1M Tris-HCl (pH 8.0). The eluted IgG was dialyzed overnight againstPBS, and the protein content determined by measuring the absorb-ance spectrophtometrically at 260 nm and 280 nm and extrapolat-ing from a standard curve.

Preparation of hormone-free mithun plasma

For the preparation of oxytocin-free mithun plasma, blood sam-ples were collected from old, non-lactating, non-pregnant,anestrous mithuns in which the oxytocin concentration in the bloodwas anticipated to be at a bare minimum. Necessary precautionswere taken to ensure that the animals were not stressed during thecollection of blood. After centrifugation of the blood, the plasma wastreated with a charcoal and dextran mixture to remove any traceamount of oxytocin in the plasma, as follows. Activated charcoal (14g) and dextran T-70 (1.4 g) were mixed with 100 ml plasma, andthe mixture was washed with distilled water by thorough mixing witha magnetic stirrer overnight at room temperature. After allowing themixture to stand for 5 min, the supernatant was discarded. The mix-ture was subjected to the washing process three more times at 8-hintervals to remove all traces of suspended fine charcoal particles.Mithun plasma was then added to the washed charcoal-dextranmixture and mixed thoroughly for 2 h at 4

°

C. The mixture was cen-trifuged at 3,000 rpm for 1 h at 4

°

C, and the supernatant was fil-tered to remove suspended particles. The filtrate was again centri-fuged at 10,000 rpm for 1 h at 4

°

C, and the supernatant was re-filtered. This oxytocin-free plasma was then stored at –20

°

C forfuture use.

Preparation of biotinyl-oxytocin conjugate

An amount of 1 mg of oxytocin (Saxon Biochemicals, Han-nover, Germany) dissolved in 1 ml of PBS (50 mM NaPO

4

, 0.15 MNaCl) was added to 5 mg of biotinamidedocaproate-N-hydroxysuc-cinimide ester (biotin; Sigma, Germany) dissolved in 50

µ

l of dime-thyl sulfoxide. The reagents were vortexed and then incubated forhalf an hour. To separate the biotinyl oxytocin from free biotin and/or any unbound oxytocin, the mixture was loaded on a small Sepha-dex G-25 gel-filtration column (PD-10; Pharmacia, Sweden). Thecolumn was then eluted with 30 to 40 ml PBS and fractions of 1 ml

Mithun Oxytocin Immunoassay 635

were collected and the protein peak estimated by UV spectropho-tometer readings at 260 and 280 nm. The fractions correspondingto the first peak containing the purified biotinyl-oxytocin conjugatewere pooled, and 1 ml of 1% bovine serum albumin (BSA) in PBSwas added. For long-term preservation at –20

°

C, the conjugate wasmixed with an equal volume of glycerin to prevent freezing andstored in 1-ml aliquots.

Oxytocin antibody and bovine oxytocin standards

An oxytocin-specific antibody raised in rabbits against oxytocin-thyroglobulin was used (generously supplied as a gift by Prof. Dr.W. Wuttke, Frauenklinik, Goettingen, Germany). The antiserumused in this assay was specific for oxytocin and did not cross reactwith any other related octapeptides tested, namely lysine-vaso-pressin, arginine-vasopressin, mesotocin, lysine-vasotocin, andarginine-vasotocin (<0.001%). The bovine oxytocin standards usedin the assay were purchased from Saxon Biochemicals, Hannover,Germany.

EIA Procedures

First coating, second coating, washing

The first coating was performed by adding 0.63

µ

g of goat IgGto rabbit IgG dissolved in 100

µ

l of coating buffer (15 mM Na

2

CO

3

,35 mM NaHCO

3

, pH 9.6) per well of a microtiter plate (GreinerLabotechnik). The plates were subsequently incubated overnight at4

°

C. For blocking the remaining binding sites, 300

µ

l of 1% BSA inphosphate buffer were added to all wells, and the plate was incu-bated for 40 to 50 min at room temperature under constant shaking.Each well was washed twice with 350

µ

l of washing solution (0.05%Tween 20) using an automated microtiter plate washer (Model ELx50, BIO-TEK Instruments, Vermont).

Assay protocol

Duplicates of 200

µ

l of unknown plasma or standards preparedin hormone-stripped charcoal dextran-treated plasma ranging from0.1 to 100 pg per 200

µ

l were simultaneously pipetted with the aidof a diluter dispenser into respective wells, along with 100

µ

l of oxy-tocin antibody diluted 1:400,000 in assay buffer (50 mM NaPO

4

,0.15 M NaCl, 0.02% thimerosal, pH 7.4) supplemented with 10%oxytocin-free mithun plasma. Plates were incubated overnight atroom temperature. They were then decanted and washed fourtimes with washing solution before addition of 100

µ

l of biotinyl-oxy-tocin-conjugate diluted 1:400,000 in assay buffer. The plates werefurther incubated for 30 min, decanted, and washed twice. Then 20ng streptavidin-peroxidase (Sigma, Germany) in 100

µ

l of assaybuffer were added to all the wells, and the plates now wrapped inaluminium foil were incubated for a further 30 min with constant agi-tation. All steps were performed at room temperature.

Substrate reaction

The plates were washed four times with washing solution andincubated another 40 min in the dark after the addition of 150

µ

l ofsubstrate solution per well (substrate buffer=0.05 M citric acid, 0.11M Na

2

HPO

4

, 0.05% ureum peroxide, pH 4.0 adjusted with 5N HCI;substrate solution=17 ml substrate buffer containing 340

µ

l3,3´,5,5´-tetramethyl benzidene and 12.5 mg/ml dimethyl sulfoxide;Sigma, USA). The reaction was stopped by the addition of 50

µ

l 4N H

2

SO

4

, and the color was measured at 450 nm with a 12-channelmicrotiter plate reader (Model ECIL, Microscan, India).

Parallelism between bovine oxytocin standards and endogenousoxytocin in mithun plasma

Homology between the bovine oxytocin standards used andendogenous oxytocin in mithun plasma was assessed by conduct-ing a parallelism test. For this purpose, four mithun plasma samples(200

µ

l) from four different animals containing high concentrationsof endogenous oxytocin were serially diluted with EIA assay buffer

(containing 3.1, 6.3, 12.5, 25, 50,100, and 200

µ

l mithun plasma)and assayed along with the bovine oxytocin standards (in buffer).

Statistical analyses

Means and standard errors of means of hormone levels werecalculated for each sequential blood sample. The data on hormonalconcentrations were analysed by an ANOVA for repeated mea-sures, with a post-test for linear trend to compare hormonalchanges during different days and across time using InStat software(Graphpad, San Diego, USA). To statistically assess the parallelismbetween standard and endogenous hormones, a non-linear regres-sion test was performed using Prism 4.01software (GraphPad, SanDiego, USA).

RESULTS

Enzyme immunoassay (EIA) of oxytocin

Titration of biotinyl-oxytocin and oxytocin antiserum

A two-dimensional titre determination was performed.Antibody dilutions ranging from 1:50,000 to 1:1,600,000 andbiotinyl-oxytocin dilutions of 1:50,000 to 1:3,200,000 weretested. The antibody titre of 1:400,000 and the biotinyl-oxy-tocin conjugate titre of 1:400,000 were found to be optimumand achieved an OD

450

of around 1.4–1.3.

Assay sensitivity, precision, and specificity

To determine the possible interference of plasma withthe assay sensitivity, oxytocin standards in various amountsof mithun plasma (50, 100 and 200

µ

l) were run in an assay.While the absolute binding sensitivity did not vary withincreasing plasma volumes, there was a drop in the OD

450

at higher plasma volumes (Fig. 1). On account of this inter-ference, all standards ranging from 0.1 to 100 pg/200

µ

l/wellwere subsequently prepared in hormone-free plasma andwere run along with the unknown in the test. All assays werehence conducted using duplicate 200

µ

l aliquots of unknownplasma samples and standards in 200

µ

l volumes per well.The lowest detection limit was 0.1 pg per well, which corre-sponded to 0.5 pg/ml plasma.

Intra- and inter-assay coefficients of variation deter-mined using pooled plasma were found to be 4.4 and 6.4%,respectively.

Fig. 1.

Effect of different volumes (0, 50, 100, and 200

µ

l) ofmithun plasma on percent binding in an oxytocin standard curve. Aswith different volumes of plasma, the standards were also preparedin 20

µ

l of assay buffer. Optical density was measured at 450 nm.

M. Mondal

et al

.636

The antiserum used in this assay was specific for oxy-tocin and did not cross-react with any of the other relatedoctapeptides tested (<0.001%).

Parallelism between bovine oxytocin standards and endoge-nous oxytocin in mithun plasma

Conducting a parallelism test assessed the cross-reac-tivity between the bovine oxytocin standards used andendogenous oxytocin in mithun plasma. When increasingplasma volumes and increasing standard concentrationswere plotted, a parallel drop (P<0.05) in relative percentbinding was observed (Fig. 2). The two curves were almostparallel to each other, thereby confirming the actual oxytocinprofiles in mithun plasma.

Plasma oxytocin profile during milk ejection

Plasma oxytocin profiles before, during, and after themilking of individual animals are presented graphically inFig. 3. Before the start of udder stimulation, the levels ofplasma oxytocin were low. Within a minute after udder stim-ulation, there was a spurt of oxytocin release in all three ani-mals. In one animal (animal A, Fig. 3), the concentration ofplasma oxytocin ranged from 93–179.2 pg/ml. The levelincreased sharply immediately after the start of the milkingstimulus and reached a concentration of 369.7 pg/ml at 1.5minute after the onset of the stimulus. There were minorfluctuations in concentration thereafter until the peak con-centration of 500 pg/ml was reached 7 min after the onsetof the stimulus. Concentration decreased thereafter towardend of milking. In animal B, the concentration of plasma oxy-tocin during the pre-stimulation period ranged from 35.4–66.5 pg/ml. It increased immediately after initiation of milkingto reach a peak value of 500 pg/ml at 2 min. The leveldeclined thereafter toward the end of milking. In animal C,pre-stimulus concentrations of plasma oxytocin ranged from22.2–28.9 pg/ml. This level increased sharply to a peak levelof 480.5 pg/ml at 1.5 min after the initiation of milking. Thelevel of oxytocin was high during milking in all the animals,and fell sharply thereafter toward the end of milking. Hor-

mone concentrations then remained low until the conclusionof sampling.

Plasma progesterone and oxytocin profiles during theestrous cycle

The daily levels (mean

±

S.E.M.) of plasma progesteroneand oxytocin during the estrous cycle are presented graph-ically in Fig. 4. The differences in mean plasma progester-

Fig. 2.

Parallelism between bovine oxytocin standards and arange of serially diluted volumes (3.1, 6.3, 12.5, 25, 50, 100, and200

µ

L) of mithun plasma containing high a high level of endoge-nous oxytocin (n=4). Standards for bovine oxytocin ranged from 0.1to 100 pg per 200

µ

l per well.

Fig. 3.

Plasma oxytocin profiles before, during, and after milking inthree individual mithuns. Blood samples were collected at 1-minintervals for 5 min before the milking stimulus, at 30-second inter-vals until the end of milking, and thereafter at 1-min intervals until 5min after the end of milking.

Mithun Oxytocin Immunoassay 637

one concentrations on different days were found to be sta-tistically significant (P<0.01). Plasma progesterone was atbasal level (0.19 ng/ml) at estrous, started to rise thereafter,with a sharp increase during the late luteal phase, reacheda peak at day 15 of the cycle, and then declined rapidly from4 days before estrus to the basal level at estrus.

The mean plasma oxytocin concentrations on differentdays of the estrous cycle were also found to be significantlydifferent (P<0.001). The plasma oxytocin concentration waslow at estrus. Two peaks of oxytocin were recorded, one atday 6 and another at day 18. During the early (0–6 days),mid (7–13 days), and late (14–20 days) luteal phases, themean plasma levels of oxytocin were 106.6

±

39.1, 77.3

±

9.8,and 102.1

±

38.6 pg/ml, respectively (Fig. 5).

DISCUSSION

Standardisation and validation of oxytocin enzymeimmunoassay

The methods described here are the first report usingthe second antibody technique and the hormone–biotin–streptavidin system for mithun plasma oxytocin EIA. Theuse of the second antibody for coating the wells instead ofthe hormone-specific antibody is preferred, as it reducesassay variability associated with uneven binding of the latterto the wells and further reduces the amount of hormone-

specific antibody needed in the EIA (Meyer, 1986).The high degree of parallelism between hormone con-

centrations obtained from serial dilutions of blood samplesfrom mithun containing high levels of endogenous oxytocinand the standard curves of bovine oxytocin (Fig. 2) indicatesthat there is considerable similarity between bovine andmithun oxytocin. Without knowing the exact percentage ofcross-reactivity of the antibody with the bovine and mithunhormones, the results of the EIA can be valid in determininghormonal plasma profiles, but not true concentration values.

To obtain a high degree of sensitivity in direct EIA, lowsample volume is desirable to reduce non-specific bindingand plasma-matrix effects (Mutayoba

et al.

, 1990). Thisrequires the use of a highly specific antibody, a very efficientamplification system, and optimum ligand-antibody dilutionsat s suitable incubation temperature. In the oxytocin EIA,there was a decrease in optical density with increasingplasma volumes, although the sensitivity and the relativebinding percentage did not change (Fig. 1). In order to com-pensate for this effect, it was necessary to use the sameplasma volumes for standards and unknowns. The high sen-sitivities of the EIA procedure, (0.1 pg per well oxytocin,which corresponds to 0.5 pg/ml plasma) was sufficient tomeasure the low physiological baseline concentration ofoxytocin before milking, as well as to distinctly observehigher hormone levels during the stimulation of the udderand the milking of lactating mithuns.

The EIA procedure described here require less expen-sive instrumentation and reagents compared to RIA, andcan be adopted in developing countries where financial con-straints limit the adoption of RIA. Highly purified hormonepreparations from cattle and other species of animals areavailable, and biotinylation of hormones is not difficult com-pared to iodination procedures. Biotin and streptavidin-per-oxidase of good quality are also commercially available atrather cheaper costs and with fewer legislative regulationsthan

125

I.

Oxytocin release during milk ejection

The oxytocin profile before, during, and after milking(Fig. 3) clearly indicates the importance of udder stimulationin oxytocin release, which is known to trigger the milk ejec-tion reflex. It has been known for many years that normalwithdrawal of milk from the mammary gland during sucklingor milking involves a reflex that increases intramammarypressure. This reflex is a first-order neuroendocrine reflex,the final stage of which involves the release of oxytocin fromthe posterior pituitary, in all lactating species so far studied(Bruckmaier and Blum, 1998).

In the present study, the observation that higher levelsof oxytocin were maintained during milking indicates thatcontinual oxytocin is required to provide a contraction stim-ulus to the myoepithelial cells during milking. However, theobservation that oxytocin levels start declining just beforethe end of milking points to a possible role of the residualmilk present in the udder as well, as the relative evacuationof the alveoli in sending neural signals for decreased oxyto-cin secretion during the latter half of milking.

Milking behavior in the mithun is quite similar to that ofthe buffalo (Kumud and Prakash, 2001), but somewhat dif-ferent from that seen in cows, where the hormone level

Fig. 4.

Plasma oxytocin and progesterone profiles (mean

±

SEM) incycling mithuns (n=6).

Fig. 5.

Plasma oxytocin and progesterone profiles (mean

±

SEM) incycling mithuns during the early (days 0 to 6 of the estrous cycle),mid (days 7 to 13), and late (days 8 to 20) luteal phases of theestrous cycle in cycling mithuns (n=6).

M. Mondal

et al

.638

remains relatively high until the end of milking (Prakash

etal.

, 1998). Unlike cows and buffaloes, intermittent peaks ofoxytocin occur during milking in rats (Higuchi

et al.

, 1985).

Plasma oxytocin profile during cyclicity

In the present investigation, plasma oxytocin concentra-tion was low at estrus, and two main peaks were observed,at day 6 and day 18 of the cycle (Fig. 4). As no informationis available on oxytocin profiles during any physiologicalstage in the mithun, it is not possible to compare our resultswith other data in this species. In the profiles obtained for sixcycling mithun cows, some pulsatility was observed in oxy-tocin concentrations. A number of spikes of oxytocin duringthe luteal phase suggested a pulsatile release of the hor-mone, probably from the developed corpora lutea, asreported in cattle (Schams, 1983; Webb

et al.

, 1981; Walterand Schallenberger, 1984), buffalo (Kumud and Prakash,2001), and sheep (Flint and Sheldrick, 1982). That meanoxytocin levels were low around estrus and higher duringthe luteal phase also suggests that the corpus luteum mayperhaps be the main source of oxytocin production duringthe estrous cycle in the mithun. In contrast, peripheral bloodoxytocin was reported to be highest at pro-estrus than atother times of the reproductive cycle in the rat (Windle andForsling, 1993).

In the present investigation, the mean plasma oxytocinlevel was higher (P<0.01) during the early and late lutealphases than in the mid luteal phase (Fig. 5) of the estrouscycle. In contrast, the plasma oxytocin level has beenreported to be higher during the mid luteal phase in cattle(Schams, 1983; Walter

et al.

, 1984; Breitinger, 1984) and inbuffaloes (Kumud and Prakash, 2001). The reason forhigher plasma oxytocin levels in mithun cows during theearly and late luteal phases is not clear. A possibleluteotrophic or luteolytic role for the hormone needs to beinvestigated in detail in this species.

Interestingly, the levels of plasma oxytocin exhibited byboth lactating mithun cows around milking and cyclic ani-mals during the estrous cycle are considerably greater thanreported in any other species. The role of these greater con-centrations of oxytocin in mithuns is not fully understood.The exceptional and highly striking results of several-foldhigher levels of the hormone in the mithun may be due to itsdifferent behavior, its semi-wild status, or its social structure.Recently oxytocin has been reported to raise aggressionand lower anxiety in humans and rats (Russell and Leng,1998; Resnick, 2005) and to play a role in the maintenanceof relationships (Trinkl, 1999).

CONCLUSION

A very simple and direct enzyme immunoassay proce-dure has been developed for the first time to determineplasma oxytocin levels in mithuns. These methods areespecially important in the context of endocrine research inlaboratories located in very remote areas, where access toradiochemicals and other reagents normally required for theconventional radioimmunoassay procedure is difficult. Apartfrom the major advantage of being non-radioactive, the EIAprocedure described here also uses a highly stable biotina-lyted-hormone which has a shelf life of several years, longerthan the short shelf life of the iodinated tracer used in RIA

procedures. The EIA procedure is particularly useful for lab-oratories with limited financial resources to conduct endo-crine studies. The short duration of the assays, whichrequire no prior extraction of the samples, is an addedadvantage over conventional RIA procedures. The profile ofoxytocin during the milk ejection process shows that theendocrine mechanisms for milk ejection in the mithun couldbe qualitatively very similar to those seen in other lactatingruminant species, but quantitatively several-fold higher thanin any other species reported so far. The low concentrationsof plasma oxytocin at estrus and the two peaks at days 6and 18 of the cycle indicate that the corpus luteum is prob-ably the main source of the hormone in this species.

ACKNOWLEDGMENTS

We are grateful to Prof. Dr. W. Wuttke, Frauenklinik, Goettin-gen, Germany, for the generous gift of the highly specific antiserumagainst oxytocin. The authors also wish to thank the Director,National Dairy Research Institute, Karnal-132 001 (Haryana), Indiafor providing part of facilities used for the present work.

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(Received December 20, 2005 / Accepted April 8, 2006)