Tocolysis in VeterinaryReproduction

A post graduate credit seminaron

Speaker: DHIREN B.BHOIReg.No.-04-00303-07

VOBG-900

Major Guide:Dr. V. K. Sharma

Minor Guide:Dr. J. N. Mistry

DEFINITION

Reduced uterine contractions following administration ofmyometrial relaxing agents: Tocolytic drugs.

Tocolysis is a Greek derivative; tokos = childbirth or laborlysis = dissolution

These drugs emerged for human use and gradually linked withanimal medicine.

TOCOLYTIC DRUGS: GENERAL CONCEPT

Innes and Nicherson, 1975; Putnam et al., 1985

Smooth muscles innervated by the sympathetic nervous system:α and β receptors α receptors:- Muscular contractions

β receptors:- Relaxation

β1:confined to heart and small intestine

β2:vascular smooth muscle, myometrium, bronchial tree

So, drugs that produce this effect are termed uterine myorelaxants, tocolytic drugs or simply tocolytics.

Stimulation of β2 receptor cause bronchodilation, relaxation of

the uterus and vasodilation in most species.

Zerobin and Kondig,1980

DRUGS USED AS TOCOLYTICS

β-sympathomimetics – e.g. Isoxsuprine, Ritodrine, Terbutaline, salbutamol, Orciprenaline, Fenoterol, Clenbuterol, Meluadrine Tartrate, KUR-1246. Calcium antagonists – e.g. Nifedipine, Verapamil, Nicardipine, Nitrendipine. Oxytocin antagonists – e.g. Atosiban and all Beta-sympathomimetics. Prostaglandin antagonists – e.g. Indomethacin Other agents – e.g. glyceryl trinitrate, Extracts of Bryophyllum pinnatum, omega-3 long chain Polyunsaturates and Magnesium sulfate.

Norwitz et al., 1999

Kauppila et al., 1978, Schenken et al., 1980

Subsequently new agents invented that stimulate β2 receptors andused as tocolytics without side effects,

RitodrineSalbutamol, Terbutaline, Clenbuterol, Orciprenaline.

For examples

Isoxsuprine lactate was 1st agents to be used for treatment of labor.

β-SYMPATHOMIMETICS

Tocolytic agents

Vamerzani et al., 1996, Garg et al., 2004

Sr. no. Proprietary name Ingredient Pharmaceutical Company1 A. Duphaspasmin

B. Duvadilan

Isoxsuprine lactate Philips-Duphar (Holland)

Duphar Interfran, Bombay (India)

2 Yutopar Ritodrine chloride Philips-Duphar (UK)

3 Brethine Terbutaline Sulfate

Novartis Pharmaceuticals

(USA)

4 Ventolin Salbutamol

hemisulfate

Allen & Hanbury's Pharma.

(Canada)

5 Alupent Orciprenaline sulfate

Boehringer-lngelheim (Canada)

6 Berotec Fenoterol

bromide

Boehringer-lngelheim

(Canada)

7 Ventipulmin, Planipart Clenbuterol

hydrochloride

Boehringer-lngelheim

(Canada, Germany)

Major side effects: Cardiovascular complications

COMMERCIALLY AVAILABLE TOCOLYTIC DRUGS

MECHANISM OF ACTION

Drug administration

Adenyle cyclase activation

ATP cAMP

(www.Drug.com)

Drug transportation through carrier protein or diffused osmosis; within 10-15 min.

Protein kinese stimulation

Protein phosphorilation

Intracellular calcium sequesterization

Deminished contractile protein activity

Uterine myometrial relaxation

PHARMACOKINETICS

Unknown in ruminants Entire intestine in monogastric animal PH affects the absorption Higher/lower PH:- Reduce absorption Neutral PH:- Increase absorption (Smith, 1998)

Peak plasma level occurs within 1-3 hr after oral dose. (Morgan, 1990)

Phase: I; Oxidation, Reduction, Hydrolysis Phase: II; Conjugation

Milk, Urine and feaces Urine:-Clenbuterol in bovine. (Smith and Paulson, 1997)

Absorption

Distribution

Metabolism

Excretion

Clenbuterol: Dave et al, 1998Terbutaline: Melanie et al, 2007

Threatened abortion / Preterm labour

Controlled calving / Nocturnal delivery To reduce neonatal morbidity and mortality in dystocia.

To aid obstetrical operations; cesarean section and fetotomy

Treatment of uterine prolapse

Embryo biotechnology

CLINICAL USE OF TOCOLYTIC DRUGS

Incidence of premature labor in animals is around 5–7 %.

Gaspar et al.., 2005; King et al., 2008

Statistics indicate that preterm birth is the leading factorcausing neonatal morbidity and mortality in animals.

Haram et al., 2003, Tucker and McGuire 2004

PRETERM LABOUR / THRETENED ABORTION

300 μg, Clenbuterol, i/m, injected in a cow for the treatmentof premature labour. The treatment induced inhibition ofuterine contractions without any side effects.

Albeck, 1981

Clinical trial

Progesterone therapy is generally advocated for the treatmentof premature labour, but it has some adverse effects: When labour pain has reached to its maximum amplitudes, the drug does not work or ineffective. It prolongs gestation length, when administered during advanced pregnancy, as a result, chances of dystocia increases due to additional weight gain of the fetus.

Tocolytic effects of KUR-1246 & Ritodrine hydrochloride onOxytocin-Induced Uterine Contraction in Pregnant Sheep.

Kiguchi et al., 2002

Efficacy is adjudged on: Cardiovascular parameters of dam. : General metabolism of dam and fetus.

Ritodrine hydrochloride(Oxytocin dose 0.77–0.94 IU/kg/min)

KUR-1246(Oxytocin dose 1.00 IU/kg/min)

Dose (μg/kg/min) uterine relaxation rate (%)

Dose (μg/kg/min) uterine relaxation rate (%)

0.1 -14.3 0.001 31.1

0.3 14.9 0.003 63.1

1.0 35.3 0.010 69.6

3.0 68.8 0.030 91.3

10.0 75.5 0.100 105.9

30.0 79.4 0.300 96.3

50% inhibition dose (μg/kg/min):-- 1.5 50% inhibition dose (μg/kg/min):-0.0024

Experimental trial

Effect of Meluadrine tartrate and ritodrine hydrochloride on maternalsystolic, diastolic and mean blood pressure in pregnant goats.

Matsuda et al., 2002

Control Oxytocin 30 min 60 min 90 min 120 min

Meluadrine tartrate treated goats (0.03, 0.1, 0.3, 1 μg/kg/min)

Systolic (mmHg)

114 ± 10 116 ± 11 115 ± 11 119 ± 11 115 ± 11 111 ± 10

Diastolic (mmHg)

69 ± 7 72 ± 7 67 ± 6 67 ± 4 62 ± 4 60 ± 5

Mean (mmHg)

84 ± 8 87 ± 8 83 ± 7 84 ± 6 80 ± 6 77 ± 6

Ritodrine hydrochloride treated goats (1, 3, 10, 30 μg/kg/min)

Systolic (mmHg)

120 ± 8 120 ± 8 115 ± 8 118 ± 10 118 ± 11 116 ± 9

Diastolic (mmHg)

72 ± 5 73 ± 4 67 ± 6 66 ± 5 61 ± 7 60 ± 6

Mean (mmHg)

88 ± 6 89 ± 4 83 ± 6 83 ± 6 80 ± 7 78 ± 7

Group Basal 5 min 15 min 30 min

Xylazine, (0.1mg/kg, i/v)

7.6 ± 1.6 25.8 ± 2.0 17.2 ± 2.0 14.8 ± 2.0

Xylazine, (0.1mg/kg,)

+Clenbuterol (4μg/kg) i/v 7.4 ± 1.7 4.2 ± 1.5 8.4 ± 2.1 7.4 ± 1.4

Xylazine, (0.1mg/kg,)

+Nifedipine (80 μg/kg) i/v 7.6 ± 1.7 2.2 ± 1.6 7.8 ± 1.1 7.0 ± 1.2

Effect of Clenbuterol and Nifedipine on xylazine-induced alterationsin the frequency of uterine contractions, 5 min-1 in adult goats.

Perez et al., 1997

Bovine , Ovine & PorcinePrediction of calving time

Physical signs:-slackening of sacrosciatic ligament, relaxation of perineum and vulva, distension of udder, vaginal mucous discharge are difficult to quantify and their development in relation to calving varied considerably. Body temperature :-Too variable to be used in prediction of parturition time (either side of 38.9 ْ C).

Ewbank, 1963

Using PGF2α / Corticosteroids:- More incidence of retained placenta (Lewing, 1985)Tocolysis:- Prediction of calving time

CONTROLLED CALVING / NOCTURNAL DELIVERY

Probability (%) of cow calving within a specified periodin relation to plasma progesterone concentration.

Parker et al., 1988

Progesterone Concentration (ng/ml)

Period before calving (Hours)

0-6 0-12 0-15 0-18 0-24

0 (Undetectable) 42 80 88 93 97

0.4 18 57 70 79 90

1.0 3 20 30 41 59

>1.5 0 0 0 1 3

300 μg, i/m, Clenbuterol suppressed uterine contractions and delayparturition in cattle on an average 5 - 8 hrs without any ill effect oncow or calf, expulsion of placenta and fertility of cow.

Ballarini et al., 1978

Clenbuterol was employed to regulate the calving time (i.e. toavert night calving) by suppressing the uterine contractions. Adose of 300μg, i.m, arrested uterine contractions for about 5 hr,in 95 cows without obvious harmful effect.

Ballarini, 1978

Terbutaline,5mg/kg, i/m, has potential as a tocolytic drug to delayparturition and for various bovine obstetrical maneuver.

Melanie et al., 2007

FACTORS AFFECTING TOCOLYSIS

Stage of labour

Parity of animal

Cervical dilatation & fetal position

Pelvic area of dam

18 cross bred cows used in a trial and found that duration ofdelaying calving in the first stage of labour was much morecompare with the second stage of labour.

Group Particular Delay in Parturition

I Control (No treatment) ------

II Clenbuterol 300 μg in first stage of labour 7-10 hours

III Clenbuterol 300 μg in second stage of labour 2-3 hours

Vamerzani et al., 1996

Once cervix is fully dilated or fetal feet are passing into cervical areaClenbuterol will only delay labour for maximum of a few hours.

Arbeiter and Holler, 1980

Animal (No.)

Cervix

Degree of dilation at treatment (cm.)

Minutes to Tocolysis

(Minutes)

Duration of Tocolysis

(Hours)

Duration of parturition

after resumption of labour (Hours)

Retension of

Placenta (No.)

2-4 cm 4 cm

Heifers - 25 19 6

Mean 17.18 Mean 4.9 0.74

2Min 10.00 Min 3.5 0.5

Max 25.00 Max 7.5 1.5

Cows - 7 1 6

Mean 21.44 Mean 4.21 0.49

0Min 15.00 Min 2. 50 0.25

Max 35.00 Max 6.00 0.75Zerobin and Kundig, 1980

Clinical observations of Clenbuterol on nocturnal delivery

300 μg, Clenbuterol Hydrochloride, i/v, was used to postponedparturition in cows.

Clenbuterol 300μg, i/m, was used in heifers for postponement ofparturition.

Influence of Clenbuterol on the length (minutes) of stage-I ofparturition in heifers with large and small pelvic areas.

Control Clenbuterol

Large Pelvic areas

Heifers (No.) 10 13

Average 119 minute 468 minute

Range 30 - 230 minute 75 – 1350 minute

Small Pelvic areas

Heifers (No.) 10 13

Average 130 minute 381 minute

Range 30 - 330 minute 30 – 900 minute

Putnam et al., 1985

Influence of Clenbuterol on the length (Minutes) of stage-II ofParturition in heifers with large and small pelvic areas.

Control Clenbuterol

Large Pelvic areas

Heifers (No.) 10 13

Average 86 minute 107 minute

Range 20 – 150 minute 15 - 145 minute

Small Pelvic areas

Heifers (No.) 10 13

Average 120 minute 54 minute

Range 45 - 270 minute 5 - 240 minute

Putnam et al., 1985

Clenbuterol 300 μg, i/m is useful to postpone the parturition atdifferent stages in cows.

Group Cows Average interval from injection to calving

Group-I (2 fingure cervical dilation) 32 23.4 hours

Group-II (4 fingure cervical dilation) 40 14.2 hours

Group-III (full hand Cervical dilation) 19 9.9 hours

Group-IV (Fetal parts in cervix) 9 5.2 hours

Greene, 1981

During Clenbuterol treatment in cows, the duration of tocolysisdepends on the position of the fetus at the time of treatment, beinglongest at the beginning of the labour process (Stage-I).

Greene, 1981

Clenbuterol treatment in ewes effectively resulted in to delayedparturition for at least 10 hr. in most ewes.

Plant and Bowler, 1988

Clenbuterol, 240 μg, i/m, abolished uterine motoricity for 8-10 hrssuspending overnight lambing in 91% of ewes subjected to trial.

Delatour and Roizard, 1979

OVINE

Nifedipine 80 μg/kg, i/v given to sheep and found that it delayedthe parturition for 6-7 hours.

Parez et al., 1997

Clenbuterol, 6 ml, i/m given to ewes to delay lambing for 7 hours.

Hirst et al., 2005

Clenbuterol had been used in 13 pigs for postponement ofparturition at dose rate of 150 μg, i/v (Planipart).

Interruption of parturition Nocturnal delivery

Suitable for all the phases of farrowing,

Interrupts labour for several hours,

Results into unhindered farrowing without affecting piglet vitality.

PORCINE

Zerobin and Kundig, 1980

Interruption of parturition

Gilts (No) No. of piglets already born

Onset of

Action

(min.)

Duration of Tocolysis (Hours)

Duration of parturition after

resumption of labour (Hours)

No. of piglets born (Stillborn)

7 13 Mean 8.28 2.83 2.14 7 – 14 (0-3)

Min 5.00 2.5 1.5

Max 12.00 3.25 2.5

Postponement of parturition

Gilts (No.) Duration of postponement (hrs)

Duration of parturition (Hours)

Number of piglets born

Alive Stillborn

5

Mean 14.89 3.0

7 - 12 0 – 1Min 12.5 2.5

Max 17.0 -

Zerobin and Kundig, 1980

DYSTOCIA

Tocolytic drug Dose & Species

Tocolytic effect within

Duration of tocolysis Reference

Isoxsuprine lactate

5-10 ml: Cow & Mare

10-15 min. 1-1.5 hours

Horvath and Bacsfay, 1981

Erkert and

Macallister, 2002

1-2 ml: Ewes

1-4 ml: Saw

0.2-1 ml: Bitch

0.1 ml: Cat

Clenbuterol hydrochloride

300 μg: Cow 15-30 min 1.5-2 hours Menard, 1994

300 μg: Cow 15-30 min 1.5-2 hours Jonker et al., 1991

100 μg: Sheep

10-15 min. 1-1.5 hours Zerobin and Kundig, 1980

300 μg: Mare 10-15 min. 1-1.5 hours Riepe, 1981

ADVANTAGES OF TOCOLYSIS IN OBSTETRICAL MANEUVER

Less requirement of epidural anaesthesia

Easy and correct diagnosis of obstetrical defects

Facilitated obstetrical maneuvers Repositioning of head and neck deviations. Corrections of malpresentations and malpostures Repulsion and rotation of fetus Correction of forelimb retension, hock flexion, and breech presentation

Low incidence of retained placenta (Menard, 1994)

Low incidence of genital prolapse (Albeck, 1981)

REQUIREMENT OF EPIDURAL ANAESTHESIA

300 μg, i/m, Clenbuterol given to cows in dystocia cases

Number of animals Epidural anaesthesia

Treatment 219 80 (37%)

Control 456 319 (70%)

Menard, 1994

Results of Clenbuterol, i/v, 0.6-0.8 μg/kg body wt. before 15-20 minof obstetrical corrections in cow.

Dystocia Total Tocolysis

Fetal

Very good

Oversize 6

Sacro-pubic position 35

Transverse presentation 2

Forelimb Retension 25

Head Deviation 41

Breech presentation 41

Hock Flexion 5

Maternal

Very good Uterine Torsion 70

Delayed cervical dilation 7

Total 232

Menard, 1994

Incidence of retension of placenta was lower in dystocia casestreated with Clenbuterol compared to non-treated cows.

Dystocia Retension of Placenta Total

Yes No

Treated 48 (20.68%) 184 (79.32%) 232

Control 150 (32.89%) 306 (67.11%) 456

Total 198 490 688

Influence of Clenbuterol treatment on Retension of placenta

Menard, 1994

CAESAREAN SECTION

Advantages

Facilitate veterinarians’ work.

Easy extraperitoneal lifting of the uterus. (Hassett and Sloss, 1984) No risk of anaesthetic induced recumbancy. Impermeable closure of uterine muscles. (Horvath and Bacsfay, 1981) Easy suturing of uterus and no incidence of ROP. (DeNooij, 1984) Prevent post operative adhesions.

Reduce mortality of dam and calf. (Menard and Diaz, 1987)

Cows treated with Clenbuterol 0.6 μg/kg body wt. 15-30 minprior to caesarean, then good proportion of exteriorization wasnoticed than in the non-treated animals.

Menard and Diaz, 1987

Caesareans

Uterine Exteriorization

TotalYes No

Treated (n=63) 54/63 (85.7%) 9/63 (14.3%) 63

Non-treated (n=90) 27/90 (30%) 63/90 (70%) 90

Total 81 72 153

The incidence of retension of placenta and loss of cows and calfalso noticed very less in caesarean performed in cows usingClenbuterol, 0.6 μg/kg body wt.

Menard and Diaz, 1987

Treated Group (n=63) Non-treated Group (n=90)Retension of placenta

Uterine Adhesions

Deaths Retension of placenta

Uterine Adhesions

Deaths

Cow Calf Cow Calf

(8/63) 12.7%

(11/63)

17.46%

(5/63) 7.94%

(4/42) 9.52%

(38/90) 42.2%

(44/90)

48.88%

(14/90) 15.56

%

(8/57) 14.04

%

Good plane of uterine relaxation observed in cows affected with dystocia treated with 5 mg/kg, i/v, terbutaline.

Melanie et al., 2007

Uterine Exteriorization

Total animals Exteriorization achieved No response

Treatment Group Control Group

66

90%

59

10%

760%

40

40%

26

66

10 ml Isoxsuprine, i/m, given in cows during caesarean section

Ahlers and Anderson, 1967

A cesarean section performed in a Berkshire saw treated with6 ml Isoxsuprine, i/m, a good uterine relaxation was noticedand uterus could be easily exteriorized.

Narasimhan and Thangaraj, 1969

Very good uterine relaxation, improved ease of manipulationand exteriorization of uterus was noticed during Caesareanperformed in Sheep (n=3) using Clenbuterol, 0.8 μg/kg b. wt.

Menard and Diaz, 1987

Sheep

Pig

FETOTOMYConditions Drugs Animals Doses &

route

Uterine Relaxation

Reference

Fetal Postural defects

Isoxsuprine Cows 10 ml, i/m Very good Horvath and Bacsfay, 1981

Clenbuterol Cows 300μg, i/m

Very good DeNooij, 1984

Clenbuterol equine 75μg, i/m Very good DeNooij, 1984

Clenbuterol Cows 300μg, i/m

Very good Menard and Diaz, 1987

Fetal Emphysema

Isoxsuprine Cows 10 ml, i/m Very good Horvath and Bacsfay, 1981

Isoxsuprine Cows 10 ml, i/m Very good Narasimhan and Thangaraj, 1969

UTERINE PROLAPSE

Animals Drug Doses

&

route

Onset of

action

Clinical traits of uterine prolapse Reference

Reposition Reoccurrence

Cows Clenbuterol 3 mg, i/m 15 min. Achieved No Murling, 1983

Isoxsuprine 10 ml, i/m 15 min. Achieved No Narasimhan and Thangaraj, 1969

Clenbuterol 0.3 mg, i/m

15 min. Achieved No Albeck, 1981

Clenbuterol 0.3 mg, i/m

10-20 min

Achieved No DeNooij, 1984

Buffaloes Isoxsuprine 10 ml, i/m 15 min Achieved No Narasimhan and Thangaraj, 1969

Sheep Clenbuterol 0.001mg/kg,i/m

15 min Achieved No Zennetti, 1983

Kind of animal Doses Onset of action

Non-descript cow-2 30 mg 25 minutes

Non-descript cow-1 60 mg 20 minutes

Non-descript cow-1 100 mg 15 minutes

Cross-bred jersey cow-1 100 mg 25 minutes

She buffalo-1 50 mg 20 minutes

She buffalo-1 100 mg 15 minutes

Rajasekaran et al., 1980

Seven cases of bovine uterine prolapse were treated with uterinerelaxant Isoxsuprine with varying doses from 30-100 μg i/m,according to size of the animal and good results were achieved.

UTERINE TORSION

Animal Drug

Doses &

route

Onset of action

Uterine relaxation Detorsion Reference

Cow

Isoxsuprine 10 ml, i/m 15 min Good Achieved Horvath and Bacsfay, 1981

Clenbuterol 0.3 mg, i/m 20 min Good Achieved Albeck, 1981

Clenbuterol 0.6μg/kg, i/v 15 min Good Achieved Menard, 1994

Recipients received 300μg Clenbuterol, i/m, 30-90 min. prior to Embryo transfer. Pregnancy rates varied from 19-56 % in Clenbuterol treated cases.

The results indicate no improvement in pregnancy rates. But treatment resulted into a relaxant effect on uterine myometrium.

Wenkoff, 1986

Clenbuterol used to relax non-pregnant uterus & increased pregnancyrates in cattle recipients undergoing surgical embryo transfer.

Coulthard, 1982

EMBRYO TRANSFER

Surgical method

Non-surgical method

In a field trial, Clenbuterol, 364 μg (10 ml) was given by i/m,immediately following the embryo transfer in cattle,

No Transfer Clenbuterol Control Total

1 Surgical 30/49 62.5 % 24/47 51.0 % 54/95 56.8 %

2 Non-surgical 57/97 58.7 % 58/98 59.2 % 115/195 58.9 %

3 Non-surgical 47/92 47.1 % 41/87 51.1 % 88/179 49.2 %

4 Surgical 31/68 45.58 % 28/71 39.43 % 59/139 42.4 %

Total 163/305 53.4 % 153/303 50.5 % 316/608 51.9 %

Recipient Pregnancy Rate

Barnes and First, 1985

The effects of Planipart (Clenbuterol), Duvadilan (Isoxsuprine)and Aerolin (Salbutamol) were tested on pregnancy rates of cowsreceiving the embryos transferred by non - surgical method.

Gregory et al., 1986

Drug Dosage Transferred Embryos Pregnancy

Control ------ 38 N=15 39 %

Clenbuterol (Planipart) 0.3mg 34 N=13 38 %

Isoxsuprine (Duvadilan) 20 mg 77 N=37 48 %

Salbutamol (Aerolin) 4.8mg 24 N=11 46 %

Total 300 μg Clenbuterol given i/m to cattle recipients at the timeof ovarian palpation, 30 - 180 min. prior to embryo transfer hadeffectively improved pregnancy rate after surgical embryo transfer.

Group Number of animals

Pregnancy Rate

Control 344 45%

Treatment 264 55%

Group Number of animals

Pregnancy Rate

Control 213 51%

Treatment 307 49%

SurgicalEmbryo Transfer

Maplotoft et al., 1986

Non-surgicalEmbryo Transfer

Among the β-sympathomimetic drugs used in reproduction, onlyClenbuterol and Isoxsuprine have been used widely in clinicalmanagement of obstetrical disorders apart from embryo biotechnology with encouraging post therapeutic results.

CONCLUSIONS

The efficacy of these drugs is mostly assessed clinically andpharmacokinetic of each needs to be studied in detail to ensure wideuse with awareness of adverse effects of drug metabolites, if any.