ISSN 1062-3590, Biology Bulletin, 2008, Vol. 35, No. 6, pp. 615–618. © Pleiades Publishing, Inc., 2008.Original Russian Text © V.V. Rozhnov, I.E. Chernova, S.V. Naidenko, 2008, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2008, No. 6, pp. 713–717.

615

INTRODUCTION

Many problems in sable (

Martes

zibellina

) farmingare accounted for by specific features of its reproduc-tive biology and, in particular, by a long embryonic dia-pause. Since there are no reliable methods of pregnancydiagnosis in the sable, the probability of offspring pro-duction remains indeterminate, resulting in increasedexpenditures for the maintenance of sables in farms.

Sables mate in late June to early August, giving birthto the offspring in April. After mating, the developmentof the fertilized egg is suspended at the blastocyst stagefor more than seven months, with implantation takingplace only 30–45 days before delivery (Manteifel’,1934; Starkov, 1947; Polyntsev, 1975). Thus, preg-nancy in the sable includes two periods: the embryonicdiapause and the postimplantation period (true preg-nancy), with the former being markedly longer than thelatter. Therefore, approaches to pregnancy diagnosis inthese periods are different.

Female sables have induced ovulation, which takesplace 72–84 h after successful mating (Bernatskii et al.,1976); yellow bodies formed in place of ovulated eggsproduce progesterone, being the main source of thishormone in the female body until implantation. As inmost mammals, pregnancy in the sable is accompaniedby significant changes in plasma levels of steroid hor-mones, which may serve as its reliable indicator: bloodprogesterone in pregnant females is increased, includ-ing that during the diapause. Plasma estrogens duringthe diapause are also 2.5–3 times higher than in non-mated females. During true pregnancy (after implanta-tion), progesteron and estrogen levels further increasedue to their synthesis in the placenta (Polyntesev and

Volchek, 1975; Polyntsev et al., 1977; Polyntsev,1980). However, pregnancy diagnosis by blood hor-mone levels in sables is impossible because of theirhigh vulnerability to stress: animal capture and bloodsampling may result in embryonic mortality and spon-taneous abortion.

An alternative approach to pregnancy diagnosis isnoninvasive monitoring of hormones by methodswidely used in studies on other species of carnivores,including measurements of progesterone metabolites inexcretions (primarily urine and feces). The concentra-tion of these metabolites during pregnancy is known toincrease by factors of 25 to 100 (Brown et al., 1994),decreasing to the baseline level soon after the birth ofthe offspring. Changes in the level of progesterone orits metabolites in excretions of female carnivores allowthe diagnosis of not only pregnancy itself but also ofovulation (Brown et al., 1994, 2001; Spanner et al.,1997; Moreira et al., 2001). However, such studies werenever performed in mustelids with a long diapause inembryonic development, such as the sable. The methodof pregnancy diagnosis in this species by the level ofsteroid hormones in urine (Polyntsev et al., 1977) iseffective but extremely labor-intensive and, therefore,inapplicable in fur farming.

To develop a method of pregnancy diagnosis in thesable on the basis of noninvasive methods of hormonestatus assessment, we made measurements of immu-noreactive compounds (IRC) binding with antibodiesto progesterone in excrements of female sables at dif-ferent stages of the reproductive cycle and estimatedthe parameter most suitable for diagnostic purposes.

Approaches to Pregnancy Diagnosis in the Sable (

Martes zibellina

, Mustelidae, Carnivora) by Noninvasive Methods: Postimplantation Period

V. V. Rozhnov

a

, I. E. Chernova

b

, and S. V. Naidenko

a

a

Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 119071 Russiae-mail: rozhnov@sevin.ru

b

Research Institute of Fur Farming and Rabbit Breeding, ul. Trudovaya 6, Rodniki, Moscow Region, 140143 Russia

Received February 29, 2008

Abstract

—To develop a reliable approach to pregnancy diagnosis in sables based on noninvasive methods ofhormonal status assessment, the concentrations of immunoreactive compounds (IRC) binding with antibodiesto progesterone have been measured in the feces of females at different stages of the reproductive cycle. Theresults show that this concentration is higher in truly pregnant than in mated but nonreproducing females. Anincrease in the IRC concentration relative to its individual baseline level may be regarded as a reliable indicationof true pregnancy.

DOI:

10.1134/S1062359008060083

ZOOLOGY

616

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ROZHNOV et al.

MATERIALS AND METHODS

The study was performed in the OOO PushkinskiiFur Farm in May and June 2006. Sixteen females suc-cessfully mated in 2005 were included in analysis;11 of them produced the offspring in April 2006. Sam-ples of excrements were taken in different periods: dur-ing true pregnancy (March 22–25,

21.8

±

1.7

daysbefore delivery), three days after delivery (April 5–21),and between the end of lactation and the beginning ofthe next mating season (June 6–7). The IRC level wasassumed to decrease significantly after delivery, and theIRC level after the end of lactation was taken as thebaseline level. Fresh excrements were collected fromunder individual cages of sables in sheds, transferred toa freezer, and stored before analysis at

–18°

C.The extraction and measurements of IRC were per-

formed at the Chernogolovka Research Station ofSevertsov Institute of Ecology and Evolution, RussianAcademy of Sciences. The standard extraction proce-dure was used (Jewgenow et al., 2006). A 0.1-g aliquotof excrements was mixed with 0.1 g of Al

2

O

3

and 0.9 mlof 90% methanol, the mixture was vigorously shaken inan Ekros mixer (NPO Ekros, Russia) and centrifuged at4000 rpm for 10 min, the supernatant fluid was trans-ferred to a clean Eppendorf tube and diluted by halfwith distilled water, and the resulting extract was storedat

–18°

C. Before analysis, the extract was diluted200-fold with 40% methanol. The concentration ofprogesteron (IRC,) was determined by ELISA withIFA-Progesteron kits (Immunotech, Russia) and recal-culated per unit weight of fresh excrements (

µ

g/g).The data were processed statistically with the

Mann–Whitney test and Wilcoxon test for conjugatepairs. Figures in the text show mean values and theirstandard errors.

RESULTS

The average IRC level in excrements of pregnantfemales (

n

= 11) was 11.1

±

2.9

µ

g/g (Fig. 1); thatrecorded three days after delivery was slightly lower,8.9

±

2.0

µ

g/g (

n

= 11;

T

= 27,

Z

= 0.62; ns); and thebaseline IRC determined in the first ten-day period ofJune was 4.3

±

2.1

µ

g/g (

n

= 10;

T

= 9,

Z

= 1.89;

p

=

0.059). Thus, although the average IRC level in excre-ments of pregnant females was almost three timeshigher than the baseline level, the difference lacked sta-tistical significance. This could be explained in part bya high individual variation in the IRC level: in differentfemales, it varied 9.2-fold (lim 3.8–34.7) during preg-nancy, 12.8-fold (lim 1.9–23.8) immediately afterdelivery, and 137.7-fold (lim 0.15–20.6) in the repro-ductive rest period (variation in the baseline IRC level).

In the mater females that remained nonreproductive,the IRC level in March averaged

4.7

±

1.7

µ

g/g (

n

= 5)and, therefore, did not differ significantly from that inpregnant females in the same period (

U

= 16,

Z

= 1.30; ns).In addition to average parameters, we analyzed

changes in the level of IRC during pregnancy relative totheir individual baseline level. In pregnant females,IRC exceeded individual baseline levels by a factor of15.8

±

5.4 (

n

= 10). IRC levels in mated females thatremained nonreproductive were only 0.8

±

0.3 of theirbaseline levels (

n

= 4). Differences in this parameterbetween the two groups were statistically significant(

T

= 3,

Z

= 2.40;

p

< 0.05) (Fig. 2).Thus, the degree of increase in the concentration of

IRC in excrements of a certain female relative to itsindividual baseline level (that in the reproductive restperiod) could be regarded as a reliable indicator of truepregnancy in sables.

DISCUSSION

Noninvasive methods for monitoring the hormonalstatus of animals are becoming increasingly wide-spread, since they have a number of advantages. First ofall, they allow long-term monitoring (with regular, evendaily sampling) without affecting the behavior andphysiology animals. These methods are expedient instudies on the reproductive biology of mammals in cap-tivity, usually concerning rare species (Dloniak et al.,2004; Jewgenow et al., 2006), as well as in natural pop-ulations (Creel, 2001). In most cases, the concentra-tions of sex hormones (Jewgenow et al., 2006) and glu-cocorticoids (Creel et al., 1992, 1997) are measured.Difficulties in applying such methods arise due mainlyto species-specific features of the metabolism of certain

20

468

10121416

Pregnant Parous Baseline

IRC

,

µ

g/g

Fig. 1.

Average concentrations of immunoreactive com-pounds (IRC) binding with antibodies to progesterone infeces of the same female sables at different stages of thereproductive cycle.

05

10152025

Pregnant False-pregnant

IRC

act

ual/

IRC

bas

elin

e

Fig. 2.

Ratios of actual to baseline concentrations of immu-noreactive compounds (IRC) binding with antibodies toprogesterone in pregnant and false-pregnant (mated butnonreproducing) female sables (

U

= 3,

Z

=2.40;

p

< 0.05).

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APPROACHES TO PREGNANCY DIAGNOSIS 617

hormones. Steroid hormones are usually excreted in theform of metabolites and various conjugates, with thestructure of metabolites being often different even intaxonomically close species (Graham et al., 1995; Jew-genow et al., 2006). Therefore, the development of arelevant method for every new species needs physiolog-ical and biological verification (Palme, 2005; Toumaand Palme, 2005).

Noninvasive methods for the diagnosis of preg-nancy, including its early stages, by an increase in thelevel of progesterone (or IRC binding with antibodiesto progesterone) in excrements have been developed fora number of rare carnivore species for the purposes oftheir breeding in captivity. In different species, the levelof progesterone in excrements may be indicative ofovulation (Moreira et al., 2001; Brown et al., 2001) aswell as help in the diagnosis of false or true pregnancy(Spanner et al., 1997) and their differentiation fromeach other (Brown et al., 1994). The level of estriol inexcrements can also be used as an indicator of truepregnancy. This hormone is synthesized in the placentafrom dehydroepiandrosterone sulfate produced by theadrenal glands of embryos. For example, plasma estriollevel increases in pregnant women, and pregnancy canbe diagnosed by the level of estriol metabolites in urine(Dawson et al., 1983).

No noninvasive methods of hormonal monitoringhave yet been developed for the majority of mustelidspecies, although this group of carnivores includesmany rare and valuable species. There are numerousstudies on specific features of pregnancy progression inthe ferret (Mead and McRae, 1982; Carroll et al., 1985;Rose et al., 1993) and American mink (Petrova et al.,1983; Rose et al., 1986; Douglas et al., 1998), but all ofthem were performed by invasive methods involvingthe killing of animals. An increase in plasma progester-one in pregnant females was recorded in the sable(Polyntsev and Volchek, 1975), American mink(Meller, 1973), and polecat (Rozhnov et al., 2007). Theconcentrations of progesterone and estrogens in urinewere shown to rise during the first half of pregnancy inthe ferret (Mead et al., 1990). However, the results ofexperiments on the polecat (unpublished data) and seaotter (Larson et al., 2003) show that noninvasive meth-ods can also be successfully used for monitoring hor-monal activity in mustelids.

Polyntsev (1980) confirmed the possibility of preg-nancy diagnosis in sables by their hormonal status: theplasma levels of progestins and estrogens during theembryonic diapause proved to be two to three timeshigher in the females that subsequently produced theoffspring than in those that remained nonreproductive;these levels further increased during implantation andafter it, reached a peak 20–35 days before delivery, anddecreased significantly in the past 5–10 days beforedelivery. Tests for plasma progesterone allowed identi-fication of nonpregnant females in 90–100% cases.

Similar trends were also revealed in estrogen excretionwith urine (Polyntsev et al., 1977).

CONCLUSIONS

True pregnancy in the sable can be diagnosed non-invasively, by an increase in the fecal level of IRC bind-ing with antibodies to progesterone. However, the levelof progesterone itself is insufficient for reliable diagno-sis because of considerable individual variation in thebaseline IRC level. This variation, in turn, may be dueto significant individual differences in plasma progest-erone level as well as to individual features of hormoneexcretion. Only the difference between the IRC level infeces during true pregnancy and the baseline IRC levelin a given female is a reliable indicator of pregnancythat may be used for its diagnosis.

Although the results obtained in our experiments arestatistically significant, this method needs verificationand improvement. Thus, one female that showed noincrease in fecal IRC proved to be pregnant and pro-duced the offspring. To make the method more reliable,the comparison of IRC during pregnancy with the base-line level should be based on three to four samplestaken in each period of pregnancy, rather than on singlesamples (as in this study). It appears expedient to tracechanges in fecal IRC during the annual cycle in order toidentify their average, maximum, and minimum con-centrations at its different stages.

Thus, the analysis of fecal IRC binding with anti-bodies to progesterone allows differentiation betweenpregnant and nonpregnant females in the postimplanta-tion period (i.e., during true pregnancy). Early preg-nancy diagnosis (during the embryonic diapause) ismuch more difficult. Since yellow bodies are the mainsource of progesterone in the female body during thisperiod, the task is to develop approaches to the diagno-sis of ovulation in female sables.

ACKNOWLEDGMENTS

This study was supported by the basic research pro-gram “Biological Resources of Russia: TheoreticalFoundations of Rational Management” of the RussianAcademy of Sciences, (project no. IV.7).

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