Introduction

Reproductive hormones are readily available to veterinarians working with dogs and cats, both as small animal as well as large animal drugs. The increased importance of companion animals as family members has caused a differentiation of reproductive presenting complaints, from mostly castration a few decades ago to preventing and interrupting pregnancies, inducing heat, blocking the oestrus cycle, causing reversible sterility, treating uterine diseases or neutering-induced conditions or mammary hypertrophy and the like. These presenting complaints require in general a high degree of confidence with the clinical use of reproductive hormones, some of which have been improperly used for decades such as progestogens.^1,2 This paper will review the most common clinical indications and applications in small animals of prostaglandins, antiprolactinics, progestogens and estrogens.

Prostaglandins

Several prostaglandin F2alpha (PGF) compounds have been available for over 30 years as veterinary compounds for use in food animals and horses. Their luteolytic and uterotonic actions make them unique and very useful in small animals for several indications related to pregnancy termination and treatment of uterine conditions. Recently, the use of prostaglandin E1 (PGE1) products, such as misoprostol, has become popular for their uterotonic properties.

Early Pregnancy Termination in Bitches Only (Not In Queens)

The abortifacient efficacy of PGF involves induction of luteolysis, stimulation of uterine contraction and cervical dilation. Initially it was thought that these actions could be achieved using dosages of 250 mcg/kg,³ but it was later demonstrated that lower doses could be just as effective⁴. In dogs, the progesterone (P4) necessary to support pregnancy is entirely secreted from the corpora lutea throughout gestation. PGF will induce luteolysis and decrease P4 concentrations to nearly non-detectable levels very easily after day 25 or 30, but also as early as day 10 following onset of diestrus.⁵ The later in the cycle PGF is administered, the easier and more rapid the induction of luteolysis. Use of PGF requires subcutaneous administration 2 or 3 times a day, for 6–9 days or longer, until pregnancy termination can be confirmed by ultrasound.^6-9 The dosage varies depending on the type of PGF: natural PGF should be administered using a maximum dosage of 50–80 mcg/kg twice or 3 times daily (TID with 50 mcg/kg, BID with 80 mcg/kg), starting gradually with 1/3 to ½ the dose for the first day (or the first 2 administrations).^6,10 Cloprostenol should be used at a dose of 1–2.5 mcg/kg once daily and alphaprostol should be used at the dose of 20 mcg/kg twice daily. Side effects (which include emesis, salivation, defecation, urination and slight tachypnea) are dose dependent (i.e., displayed in 80% of bitches using doses of 250 mcg/kg natural PGF and only in 20% of bitches using doses of 50 mcg/kg natural PGF) and self-limiting, decreasing in intensity with repeated dosing. PGF do not appear to be working during the first half of diestrus in queens.

Late Pregnancy Termination in Bitches and Queens

Canine late pregnancy termination is generally adopted as a treatment when either a mismating was not observed, the female was not in the ovulatory phase or fertility of the male is unknown. Dosage of PGF compounds is the same as for early abortion, the only difference being that treatment must be continued until verification of efficacy by ultrasound as partial abortion of litters can occur if treatment is discontinued prematurely. With most dosages, 9 or more days may be required to complete fetal expulsion, although 5 to 7 days are usually sufficient.⁸ Cloprostenol at the dose of 2.5 mcg/kg subcutaneously, administered three times, at 48 h intervals, starting at day 30 of pregnancy shows a high efficacy in termination of pregnancy. Cloprostenol at even lower doses (1.0 mcg/kg) has been used in combination with a dopamine agonist treatment to terminate pregnancy shortly after implantation (which in bitches and queens occurs around 15–18 days after ovulation) starting around day 23 from ovulation.¹¹ Mismated queens can be treated with natural PGF at a dose of 2 mg/cat IM once a day, beginning at day 33 of pregnancy, as this will induce luteolysis and terminate pregnancy by expulsion of fetuses in pregnant cats. Side effects are milder than in dogs and included prostration, vomiting and diarrhea. Cloprostenol has been used in queens with success (and with fewer side effects than in bitches) at the dose of 5 mcg/kg in combination with cabergoline administered once daily from mid-pregnancy on.¹²

Uterine Evacuation (Including Treatment of Open Cervix Pyometra) in Bitches and Queens

The myocontracting action of PGF compounds is well known and can become useful when dealing with cases of open-cervix pyometra or late abortions with incomplete fetal expulsion.¹³ Dosages are the same as listed above. We frequently use PGF compounds combined with aglepristone (see paper on Clinical Use of Aglepristone) when late pregnant bitches are presented for induction of abortion, as such combined treatments are shorter and more efficacious. Care should be taken to make sure that the cervix is open, as causing uterine contractions on a closed cervix may cause uterine rupture or force uterine content up into the oviducts. Therefore, aglepristone is administered first and then PGF are used once cervical opening has occurred.

Other Clinical Applications

Prostaglandin F2a (PGF) products have been used also to induce parturition (albeit with a continuous infusion pump) in 57-day pregnant bitches with whelping occurring normally within 2–3 days.¹⁴ A shortening of the interestrous interval may be obtained using PGF alone or in combination with antiprolactinics. The use of a 6–10-day course of PGF starting after day 10 of cytological diestrus will achieve a complete luteolysis which will shorten diestrus and very often also anestrus. We have observed bitches in which a complete and permanent luteolysis was obtained with PGF early in their luteal phase coming back in heat after 70–90 days following the onset of previous proestrus.⁵ If shortening of the duration of diestrus is obtained with PGF and is then followed by an antiprolactinic treatment (which may shorten anestrus – see over), the interestrous interval will be substantially shortened. In male dogs, an increase in semen volume and occasionally also quality (often motility) may be obtained by administration of PGF at the dose of 100 mcg/kg 15 minutes prior to semen collection; this has resulted in an increase of 270% of total sperm numbers when compared to saline-treated controls, with no deleterious effect on refrigeration and freezing (Hess 2006). PGF may also be used to obtain an ejaculate from a reluctant or inexperienced dog.¹⁵

Prostaglandin E1 - A synthetic analogue of PGE1, misoprostol, has a strong uterotonic action and may be used to help evacuating uterine content in bitches and queens with pyometra. Misoprostol is marketed as a human compound under different trade names and is available in 200 mcg tablets. In the bitch it is administered at 10 mcg/kg BID orally (1/2 tablets/10 kg).¹⁶ It is very well tolerated and may be administered at home by clients. It has no luteolytic properties and few, if any, side effects mostly on the first day of treatment (vomiting=25% of cases; diarrhoea=30% of cases).¹⁷ It can be used in both bitches and queens as an adjunct to a PGF2a or aglepristone treatment to improve uterine contractility, or to continue causing uterine contractions once luteolysis has been accomplished, thus sparing the female the PGF2a -related side effects. PGE1 pills may also be dissolved in saline solution and administered intravaginally.¹⁸

Antiprolactinics

Prolactin (PRL) secretion by the lactotroph cells of the anterior pituitary gland is regulated by multiple neurotransmitters and hormones, with the major control mechanism being the activation of prolactin-inhibiting dopaminergic neurons in the hypothalamus. Prolactin is a major luteotrophic hormone and appears to be an absolute requirement for canine and feline progesterone secretion from day 30 after ovulation onwards.

Dopamine agonists like bromocriptine or cabergoline are ergot alkaloids, with strong dopamine D2-receptor agonist activity, and thus can reduce prolactin secretion, thereby suppressing progesterone levels. The serotonin antagonist metergoline indirectly stimulates endogenous dopamine secretion and thus can inhibit prolactin secretion as well. Cabergoline has a slow clearance, which allows for a single oral daily administration. Furthermore, its action when used at the currently accepted dosage of 5 mcg/kg is longer than 48 hours due to its particularly long (minimum 48 hours) half-life at the hypophyseal level. Bromocriptine mesylate inhibits PRL secretion during relatively short periods of time (half-life: ± 4–6 hours) and in a dose-dependent mode. In order to effectively inhibit PRL tone in a continuous fashion for therapeutic purposes, bromocriptine should be administered at least twice a day orally at doses 10–50 mcg/kg. Its lack of specificity leads to side effects on the cardiorespiratory system, causing hypotension due to vasodilatation (adrenergic type effect), or emesis due to stimulation of the chemoreceptor trigger zone (CTZ). Although its effectiveness has never been questioned, bromocriptine is not approved in most countries as an anti-PRL in small animals and its use in animals may be prosecuted in countries where veterinary antiprolactinic drugs are available. Metergoline is essentially a serotoninergic antagonist with dopaminergic agonist properties when used orally at doses of 0.1–0.2 mg/kg BID. Its shorter half-life requires at least administration twice a day. Its antiserotoninergic properties may occasionally induce central effects such as depression, nervousness, increased excitability, changes in appetite (anorexia or bulimia), psychotic effects (escaping from home, rarely aggressiveness), rarely vomition. Antiprolactinic drugs can be used in the bitch and the queen with three indications: pseudopregnancy, induction of abortion and induction of estrus.¹⁹

Pseudopregnancy

Antiprolactinics are currently considered the treatment of choice for pseudopregnancy. The antilactogenic action of both metergoline and cabergoline is well known.¹⁹ Their administration for 4–5 days at pharmacological doses is effective in treating pseudopregnancy signs and reducing milk production. Occasional failures can be dealt with by repeating the treatment protocol and extending it to 8 to 10 days, and also by administering at the same time metergoline (at the usual antigalactogenic dosage of 200 mcg/kg BID). Our current approach is to prescribe a 6–8-day treatment course as we feel that this decreases incidence of recurrence. Because of the potential presence of a daily pattern of prolactin secretion, antiprolactinic treatments should best be administered in the morning to achieve a more disruptive effect on prolactin secretion.²⁰

Induction of Abortion

The abortion induction properties of antiprolactinic drugs have been well studied for dopamine agonists (cabergoline and bromocriptine), while not as much is knownfor metergoline. Cabergoline and bromocriptine are effective in terminating pregnancy in dogs when administered at mid-gestation (as prolactin secretion starts around day 25) or later.^21,22 When administered after day 40 at oral doses of 5 mcg/kg for 5 days, cabergoline is effective in causing abortion in all treated bitches.²¹ If cabergoline administration is started earlier in pregnancy, at day 25, treatments that are effective later in pregnancy fail in most bitches and the pregnancy continues until terminated by retreatment at day 40. Combination treatment of cabergoline and prostaglandins has been used for induction of late abortion both in bitches and queen.^11,12 Also, in our experience alternating PGF and antiprolactinics on consecutive days works well and allows to reduce the dosage of PGF.

Estrus Induction

The estrus-inducing action of antiprolactinic drugs was initially thought to be due to the lowering of prolactin concentrations, but studies done at Utrecht have demonstrated that shortening of anestrus occurs irrespective of prolactin concentrations.²³ All 3 antiprolactinic products (cabergoline, bromocriptine and metergoline) have been used for oestrus induction in the bitch. Cabergoline and bromocriptine have consistently given positive results, while metergoline’s results have been more variable depending on dosage. Using low metergoline doses (0.1 mg/kg BID) from 100 days after ovulation until the following proestrus, the interoestrous interval will be significantly shortened.

We have used bromocriptine at the dose of 10–25 mcg/kg in 5 bitches with prolonged anoestrus: 4/5 came in oestrus within 13–28 days, and all 4 conceived and whelped (unpublished data). In a study on the use of cabergoline (5 mcg/kg, once daily for up to 28 days) in 9 bitches (7 rough collies, 1 Shetland sheepdog and 1 English setter) starting in mid-anestrus, fertile oestrus was induced in 10/11 cycles in 24±11 days with a reduction of the interoestrous interval of 1.8±0.2 months.²⁴ In our experience, the clinical use of antiprolactinics to induce oestrus has proven to be effective in about 70–80% of cases. Occasionally a bitch may take more than 40–50 days of treatment, which may cause the owner to get frustrated and discontinue the treatment.

Progestogens

Short- and long-acting synthetic progesterone compounds or progestogens have long been used in bitches and queens to control reproduction. The only short-acting product currently marketed in most countries of the world is megestrol acetate (MA), available as an oral formulation to determine oestrus suppression (short-term control) in bitches and short- as well as long-term suppression in queens. Long-acting progestogens available as veterinary drugs such as medroxyprogesterone acetate (MPA) and proligestone (PROL) are used for oestrus postponement (prolonged control). The mechanism of action by which cyclicity is blocked involves disruption of pituitary-ovarian communication resulting in lowered LH and FSH release and reduced concentrations of estrogen receptors in target tissues. Duration of effect depends on degree of pituitary responsiveness, which increases progressively during anestrus; therefore, early-mid anestrus treatments will be longer lasting than late anestrus treatments.

Progestogens act on all target organs of P4 such as uterus (increased endometrial growth and secretion), cervix (closure), motility of reproductive tract (decreased gamete transport), mammary glands (stimulation of growth). Other hormones affected by a (short- or long-acting) progestogen treatment include estrogens, inhibin and activin which may be decreased, growth hormone (GH) whose secretion by the mammary gland is increased, and prolactin which is inhibited during treatment and then shows a significant surge once treatment is discontinued. Insulin resistance is mediated by increased GH mammary secretion, and is particularly evident during treatment with MA. Endocrine side effects are transient and irrelevant when a young and healthy female receives a progestogen treatment with the right dosage and for an appropriate length of time. Unfortunately, overdosing has occurred many times in the second half of last century both in bitches and queens^1,2 and many of these cases have been reported in the literature and cited over and again causing a widespread fear about the use of these compounds.

On the contrary, a large amount of experimental data is available on the use of progestogens in bitches and queens, which dates back to when these animal species had to be used for approval of marketing of P4 compounds as human drugs in the 60s and 70s of last century. When reading the literature carefully, it becomes evident that all the case reports of pyometras, mammary nodules and hypertrophy, diabetes, endocrine imbalances and many others were all due to a) the use of very high dosing, b) too long treatments, or c) choice of the wrong candidate. For instance, treatment during diestrus (when endogenous P4 secretion is active) should be avoided as it could easily cause overdosing even if the correct dosage is used; serum P4 assay may be useful to identify risk patients and should be assayed also in queens due to a relevant incidence of spontaneous ovulations in this species. Diabetic patients or females with a history of irregular cycling, vulvar discharge, mammary nodules or liver/kidney insufficiency should not be treated. Pregnant females or pseudopregnant bitches should not be treated. Also, treatment during oestrus to suppress an unwanted heat should be done with caution and only with short-acting drugs for short periods of time, as progestogens side effects are likely to be amplified by previous estrogen exposure. Table 1 shows the suggested dosages of the most commonly used progestogen-based compounds in bitches and queens. The lowest effective dose should be used even if this may cause an earlier return to oestrus. Treatment durations of more than one year are probably adequate for young, healthy females, while shorter treatments should be considered for middle-age females who may have subclinical uterine or mammary conditions. The use of progestogens in older females should be discouraged.

Table 1. Suggested dosages of the 3 most commonly used progestogen compounds in bitches for the control of reproduction

The dosage for proligestone in bitches should be intended 10 mg/kg in large-size dogs and up to 33 mg/kg in medium- to small-size dogs.

Estrogens

Estrogens have always been considered as potentially dangerous drugs for small animals because of their role in inducing mammary neoplasia, endometrial hyperplasia, pyometra and bone marrow aplasia in bitches.²⁵ However, such dangerous side effects are a feature of long-acting compounds, while short-acting estrogens such as estriol are not associated with development of full (late) estrogenic effects. Estrogens have historically been used for contraception and for the treatment of urinary incontinence.

Unwanted Pregnancy

Several estrogenic compounds have been used for this purpose, but for most of them the risk of side effects has discouraged their clinical application.²⁶ As a consequence, estrogens are no longer considered a safe choice for canine pregnancy termination. Only estradiol benzoate, when given at low doses, has proven to be fairly efficacious and relatively safe. A compound with estradiol benzoate is marketed for veterinary use in mismated bitches in some European countries, which is to be administered at the dose of 10 mcg/kg SC on day 3, 5 and 7 post-breeding.²⁷ No short-term side effects have been reported following this protocol. However, in a retrospective study done in the UK, the incidence of pyometra in the 4 months after the administration of low doses of estradiol benzoate was 8.7%, whereas the incidence of that condition in a practice situation was estimated to be <2.0%.²⁸

Urinary Incontinence

Long-acting synthetic compounds, such as diethylstilbestrol, estradiol, estrone and other ester compounds, are characterized by a dangerous action on bone marrow and uterus because of their prolonged nuclear occupance time in estrogen receptors of target tissues.²⁹

The following is a list of estrogens which have been used to treat canine urinary incontinence:

• 17-b estradiol: 0.01 mg/kg MID SC/IM for 3 days
• Estradiol benzoate: 0.01–1.0 mg MID per os
• Estradiol valerate: 1.0 mg/10 kg BW
• Diethylstilbestrol: 0.06 mg MID, tapering out to 0.01 mg
• Estriol: 0.5–2.0 mg
• Conjugated estrogens: 0.02 mg/kg

Of all the estrogenic compounds listed above, estriol is the only one considered as a safe drug due to its short action, which is characterized by short nuclear occupance time and minimal metabolism following absorption. Estriol does not bind to sex-hormone binding globulin, which helps in preventing development of full (late) estrogenic effects such as endometrial hyperplasia, pyometra and bone marrow suppression. Estriol is available for human use as hormone replacement therapy for women in menopause, and is marketed in several European countries as a veterinary preparation to treat canine urinary incontinence. The current formulation is in 1.0-mg pills to be given once daily for 1 week, and then either gradually increase (in case of little or no response) or decrease (in case of a very good response), with the goal being to find the lowest possible dosage. Its efficacy after 42 days of 2.0 mg/day treatment was 85% in a multicentric clinical trial recently performed on 129 bitches in 4 European countries.³⁰ Hematological abnormalities are not observed when using dosages of 0.5–1.0 mg/day even for years, nor have they been reported in mid-term (3 months) chronic toxicity studies using 2.0, 6.0 and 10 mg doses.³¹ Vulvar swelling and male attractiveness were occasionally observed in bitches treated with estriol doses of >1.5 mg/day. Some signs of estrus can be observed in bitches administered higher dosages.³¹

References

1.  Romagnoli S. Progestins to control feline reproduction: historical abuse of high doses and potentially safe use of low doses. J Feline Med Surg. 2015;17(9):743–52.

2.  Romagnoli S, Lopate C. Reproductive endocrinology and breeding management of the bitch. In: Ettinger S, Feldman E, Côté E, eds. The Textbook of Veterinary Internal Medicine.Volume 2, Section XXII. 8th edition. Elsevier; 2017:1845–1859.

3.  Oettle EE, Bertschinger HJ, Botha AE, Marais A. Luteolysis in early diestrus bagle bitches. Theriogenology. 1988;29:757–763.

4.  Wheaton LG, Barbee DD. Comparison of two dosages of prostaglandin F2a on canine uterine motility. Theriogenology. 1993;40:111–120.

5.  Romagnoli S, Cela M, Camillo F. Use of prostaglandin F2 alpha for early pregnancy termination in the mismated bitch. Vet Clin North Am Small Anim Pract. 1991;21(3):487–501.

6.  Romagnoli SE, Camillo F, Cela M, Johnston SD, Grassi F, Ferdeghini M, Aria G. Clinical use of prostaglandin F2alpha to induce early abortion in bitches: serum progesterone, treatment outcome and interval to subsequent estrus. J Reprod Fertil Suppl. 1993;47:425–431.

7.  Feldman EC, Davidson AP, Nelson RW, Nyland TG, Munro C. Prostaglandin induction of abortion in pregnant bitches after misalliance. J Am Vet Med Assoc. 1993;202(11):1855–8.

8.  Romagnoli S, Camillo F, Novellini S, Johnston SD, Cela M. Luteolytic effects of prostaglandin F2-alpha on day 8 to 19 corpora lutea in the bitch. Theriogenology. 1996;45(2):397–403.

9.  Fieni F, Dumon C, Tainturier D, Bruyas JF. Clinical protocol for pregnancy termination in bitches using prostaglandin F2 alpha. J Reprod Fertil Suppl. 1997;51:245–50.

10.  Lange K, Günzel-Apel AR, Hoppen HO, Mischke R, Nolte I. Effects of low doses of prostaglandin F2 alpha during the early luteal phase before and after implantation in beagle bitches. J Reprod Fertil Suppl. 1997;51:251–257.

11.  Onclin K, Silva LD, Verstegen JP. Termination of unwanted pregnancy in dogs with the dopamine agonist, cabergoline, in combination with a synthetic analog of PGF2alpha, either cloprostenol or alphaprostol. Theriogenology. 1995;43(4):813–22.

12.  Onclin K, Verstegen J. Termination of pregnancy in cats using a combination of cabergoline, a new dopamine agonist, and a synthetic PGF2 alpha, cloprostenol. J Reprod Fertil Suppl. 1997;51:259–63.

13.  Hagman R. Canine pyometra: what is new? Reprod Domest Anim. 2017;52(Suppl 2):288–292.

14.  Meier S, Wright PJ. The induction of parturition in the bitch using sodium cloprostenol. Theriogenology. 2000;54(3):457–65.

15.  Hess M. Documented and anecdotal effects of certain pharmaceutical agents used to enhance semen quality in the dog. Theriogenology. 2006;66(3):613–7.

16.  Agaoglu AR, Aslan S, Emre B, Korkmaz O, Ozdemir Salci ES, Kocamuftuoglu M, Seyrek-Intas K, Schäfer-Somi S. Clinical evaluation of different applications of misoprostol and aglepristone for induction of abortion in bitches. Theriogenology. 2014;81(7):947–51.

17.  Romagnoli S, Fieni F, Prats A, Gardey L, Vannozzi I, Rota A. Treatment of canine open-cervix and closed-cervix pyometra with combined administration of aglepristone and misoprostol. In: Proceedings Congress European Veterinary Society for Small Animal Reproduction. Budapest, Hungary, 7–9 April 2006, p 287.

18.  Agaoglu AR, Schäfer-Somi S, Kaya D, Kucukaslan I, Emre B, Gultiken N, Mulazımoglu BS, Colak A, Aslan S. The intravaginal application of misoprostol improves induction of abortion with aglepristone. Theriogenology. 2011;76(1):74–82.

19.  Jöchle W, Arbeiter K, Post K, Ballabio R, D’Ver AS. Effects on pseudopregnancy, pregnancy and interoestrous intervals of pharmacological suppression of prolactin secretion in female dogs and cats. J Reprod Fertil Suppl. 1989;39:199–207.

20.  Romagnoli S, Milani C, Perin S, Ballabio R, Stelletta C, Mollo A, Gelli D. Effect of an injectable cabergoline formulation on serum prolactin (PRL) and milk secretion in early postpartum Beagle bitches. Reprod Domest Anims. 2009;44(Suppl. 2):148–151.

21.  Post K, Evans LE, Jöchle W. Effect of prolactin suppression with cabergoline on the pregnancy of the bitch. Theriogenology. 1988;29:1233–1243.

22.  Gobello C, Castex G, Corrada Y, Klima L, de la Sota RL, Rodríguez R. Use of prostaglandins and bromocriptine mesylate for pregnancy termination in bitches. J Am Vet Med Assoc. 2002;220(7):1017–9.

23.  Okkens AC, Kooistra HS, Dieleman SJ, Bevers MM. Dopamine agonistic effects as opposed to prolactin concentrations in plasma as the influencing factor on the duration of anoestrus in bitches. J Reprod Fertil Suppl. 1997;51:55–8.

24.  Rota A, Romagnoli S. One year of breeding management in a dog kennel. In: Proceedings of the 4th International Symposium on Canine and Feline Reproduction. Oslo, Norway, 29 June–1st July, 2000, page 83.

25.  Bowen RA, Olson PN, Behrendt MD, Wheeler SL, Husted PW, Nett TM. Efficacy and toxicity of estrogens commonly used to terminate canine pregnancy. J Am Vet Med Assoc. 1985;186:783–788.

26.  Teske E. Estrogen-induced bone marrow toxicity in the dog. In: Kirk R, ed. Current Veterinary Therapy: Small Animal Practice IX. WB Saunders; 1986:495–498.

27.  Sutton DJ, Geary MR, Bergman GHE. Prevention of pregnancy in bitches following unwanted mating: a clinical trial using low dose estradiol benzoate. J Reprod Fert Suppl. 1997;51:239–243.

28.  Whitehead ML. Risk of pyometra in bitches treated for mismating with low doses of estradiol benzoate. Vet Rec. 2008;162:746–749.

29.  Clark JH, Markaverich BM. The agonistic and antagonistic actions of estriol. J Steroid Biochem. 1984;20(4B):1005–13.

30.  Mandigers PJI, Nell T. Treatment of bitches with acquired urinary incontinence with estriol. Vet Rec. 2001;149:764–767.

31.  Hoeijmakers M, Janszen B, Coert A, Horspool L. Pharmacokinetics of oestriol after repeated oral administration to dogs. Res Vet Sci. 2003;75(1):55–9.