Can Non-invasive Hormone Profiling in Female Tigers (Panthera tigris) could Differentiate True Pregnancy and Pseudopregnancy

Certain critically endangered mammalian species depend on successful captive breeding programmes to thrive. To maintain the greatest possible biodiversity, breeding in captivity also necessitates the use of contemporary assisted reproductive techniques, yet understanding of reproductive physiology is frequently lacking in these species of animals. As a result, non-invasive hormone monitoring of the faeces and urine has grown in importance as a reproductive management technique.
 
The tigers (Panthera tigris) being the world’s largest cat are one of the critically endangered species which are at the verge of extinction. There are currently six known subspecies of tigers, but three have been vanished in the past 80 years. The subspecies that are currently recognized are the South-China, Bengal, Indochinese, Sumatran, Amur and Indochinese subspecies. Reproductive or sexual activities of tigers, such as mating, can take place all year round, but in some parts of the world with temperate climate, their sexual activities seem to be most prevalent from November to April. They are induced ovulators and the induction of ovulation is carried out by the copulation. Sexual receptivity of a female is only of few days and during that time, mating is very often.
 
The pseudopregnancy or false pregnancy or pseudocyesis is a syndrome which is characterized by the presence of signs of pregnancy in a non-pregnant animal. False lactation or nervous lactation are the terms which are used to refer pseudopregnancy as there is very low quantities of milk production is seen. This condition can be differentiated with true pregnancy by using hormones. In this non-pregnant luteal phase, the faecal progestin elevated for a shorter duration of approximately one-half to two-thirds of the gestation length. Relaxin and progesterone are said to be hormones of pregnancy in felines, between the two, relaxin is considered as early diagnosis factor of pregnancy in felines. The diagnosis of pregnancy in tigers by common methods as in domestic animals is quite difficult and stressful to the wild animals. The non-invasive technique uses analyses of faecal and urinary steroid metabolites for long-term assessment of ovarian activity in females, in particular, for pregnancy diagnosis and parturition prediction in captive species. It is difficult to identify a pregnant female tiger because there might be none of the morphological signs have been displayed by the animal in the earlier phase of the pregnancy. Therefore, the present study was designed to know the reproductive status of female tigers non-invasively after mating that whether underwent true pregnancy or pseudopregnancy by means of faecal and urinary hormones.

The research was carried out at Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, N.D.V.S.U., Jabalpur (M.P.). Samples were collected from Maharaja Martand Singh Judeo White Tiger Safari and Zoo, Mukundpur, Satna (M.P.), Van Vihar National Park, Bhopal (M.P.), Kamla Nehru Prani Sangrahalaya, Indore Zoo, Indore (M.P.) and Gandhi Zoological Park, Gwalior Zoo, Gwalior (M.P.) for fourteen weeks of experimental period from November 2021 to February 2022.
 
Experimental design
 
The experimental design was consisted of three groups viz. G I 03 non-bred animals of less than 9 years of age, G II 03 non-bred animals of more than 9 years of age and G III 03 animals after mating irrespective of their age. The duration of experiment was one year.
 
Sample collection and extraction
 
The urine and faecal samples of the tigress were collected in the morning between 8 and 9 am. Faecal samples were collected non-invasively from individual enclosures weekly for up to 14 weeks of pregnancy. Only fresh samples (deposited within 24 h) was chosen. Samples, about 20 g in size, were collected in individual plastic bags and immediately transferred in the vaccine storage box (to maintain the temperature for longer period of time of journey). These biological materials collected was brought to and processed at the laboratory for extraction and analysis of the samples’, extracted samples were then stored at -20°C in a deep freezer. Urine puddles was aspirated from the floor and immediately kept in the vaccine storage box, then frozen in aliquots at -20°C temperature till further analysis. Extraction of the faecal sample involves drying at controlled temperature, after drying ethanol extraction was carried out as per Dehnhard et al., (2012). The extracted samples were then analysed for estimation of hormones (relaxin; progesterone and prostaglandin F2 alpha metabolite, PGFM) by enzyme linked immunosorbent assay (Arbor Assays ELISA kit). For any changes in the behaviour of animal resulted due to any reproduction perception by the animal was monitored and recorded through CCTV (closed circuit television). 
 
Statistical analysis
 
The data obtained during experiment were analyzed by IBM SPSS-24 statistical software program using one way ANOVA in three groups.

True pregnancy and pseudopregnancy in big cats can be diagnosed on the basis of hormones and its metabolites in faecal and urine samples viz. relaxin, progesterone and PGFM. Since, a smaller number of references on the behaviour of pregnancy of tigers have been found, the references included here are on the basis of the experiences of the zoo keepers as well as from the unpublished articles. The concentration of the hormones obtained during experimental period is presented in the tables.
 
Faecal and urinary relaxin
 
The mean faecal and urine relaxin of the three groups in (Table 1 and 2) were compared for the experimental period, the least average value for faecal and urine relaxin has been found in the sixth week (870.6±125.5 pg/g) and thirteenth week (1307.7±56.4 pg/mL), whereas, highest mean value for faecal and urine relaxin was observed on the fourteenth week (1826.8±784.7 pg/g) and twelfth week (2987.7±1474.7 pg/mL), respectively. It has shown a significant change between the groups on the first, fifth, seventh, eighth, nineth, tenth, eleventh and twelfth week of the trial for faecal relaxin, in which the lowest average value was exhibited by the G I and the highest in G III. However, urine relaxin has shown a significant change between the groups on the fourth week of the trial with the highest value being exhibited by the G III (1676.7±133.2 pg/mL) and lowest in the G I (1183.2±90.1 pg/mL).





Corpus luteum is the primary source of relaxin in both pregnant and non-pregnant animals. Increased plasma level is seen during pregnancy, in which case, it is produced by the decidua and placenta its highest level attained prior to parturition. Its visible concentration observed on 4th week after mating of animal with a decline on 5th and again a rise and elevation observed from 7th week onwards. However, the reported mean baseline faecal relaxin by Harris et al., (2008) were not in agreement to our findings.
 
Faecal and urinary progesterone
 
The mean faecal and urine progesterone of the three groups (Table 3 and 4) were compared for the experimental period, the least average value for faecal and urine progesterone has been found in the fourth week (11845±2747 pg/g) and first week (1196±231 pg/mL), whereas, highest mean value for faecal and urine progesterone was observed on the fourteenth week (20239±4531 pg/g) and sixth week (4661±1143 pg/mL) respectively. No significant change has been observed between the groups for faecal progesterone, whereas, urine progesterone has shown a significant change between the groups on the nineth, twelfth and thirteenth week of the trial with the highest value being exhibited by the G III on nineth week (7517±2831 pg/mL) and lowest in the G I at same week (1163±586 pg/mL).

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The mean baseline faecal progesterone reported by Panda et al., (2017) were similar to our finding in the respective reproductive status of animal. Qaio et al., (2019) reported urine progesterone and their finding was more as compared to our findings. The significant increase in the urine progesterone observed at the respective weeks was might be due to persistence of corpus luteum (CL). The sixth week had showed a two-fold increase, whereas, the four-fold increase in the urine progesterone concentration has been observed in the twelfth and the thirteenth week. This might be due to a greater number of CL resulted from multiple mating in due course multiple ovulations and might get retained in conditions like pseudopregnancy or actual pregnancy.
 
Faecal and urinary PGFM
 
The mean faecal and urine PGFM of the three groups (Table 5 and 6) were compared for the experimental period, the least average value for faecal and urine PGFM has been found in the fourth week (8960.1±1203.6 pg/g) and second week (7110.2±1967.5 pg/mL), whereas, highest mean value for faecal and urine PGFM was observed on the thirteenth week (24579.3±6295.8 pg/g) and thirteenth week (21766.8±13574.5 pg/mL) respectively. The significant change has been observed between the groups for faecal PGFM on second and fourth week. Urine PGFM did show significant change between the groups on first and nineth week of the investigation.

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The present findings are in conjunction with the findings reported by Finkenwirth et al., (2010) and Dehnhard et al. (2012). The mean baseline faecal PGFM reported by Dehnhard et al. (2012) in Sumatran tigers differ in maximum levels of 0.4 to 2.0 µg/g faeces PGFM, whereas, the mean baseline urinary PGFM reported by Finkenwirth et al., (2010) was 1.5 ng/mL. Faecal as well as urinary PGFM concentration attain a significant rise from the baseline during the end of the pregnancy peaking towards parturition. Elevation of PGFM observed after nine weeks of gestation in big cats whereas, it is 2-4 days prior to parturition in female dogs. Unlike pregnant females, pseudopregnant and nonpregnant females does not show this elevated pattern. In addition, faecal and urine PGFM concentrations decrease drastically at the time of abortion and premature birth, indicating a strong relationship of this hormone with the maintenance of full-term pregnancy. 
 
Behavioural symptoms of pregnancy
 
The decreased frequency of scent marking has been observed in the gravid animal. The animal has been noticed that; a particular place has been selected to rest upon. Most of the time, the animal, was seen to be in recumbency and has spent time for sleeping. The enlargement of abdomen was seen in the last stage of pregnancy with protruding teats. The gradual restlessness of the animal was seen and has been recorded, the continuous avoidance of male by the female has been perceived. Later, it started search of secluded place for cubbing, so that, the cubs would be saved from any noxious deed.

Relaxin the hormone for early detection of pregnancy in felines gives elevated concentration from 49 days after mating and attains a peak during parturition, whereas, no elevation or peak is observed in pseudopregnant animals. Progesterone, in true pregnancy remains elevated from 7^th week onwards after mating. It gets dropped after 7^th week with wide fluctuation. PGFM elevation observed after nine weeks of gestation in true pregnancy whereas, no elevation will be observed in pseudopregnant tigress. Therefore, from the investigation it is concluded that the pseudopregnancy could be differentiated on the basis of hormone concentration along with the behavioural changes in the pregnant animal. Relaxin and progesterone both as early as 49 days after mating, whereas, PGFM elevation and peak at around 63 days after mating could differentiate between true pregnancy and pseudopregnancy.   

I would like to acknowledge the Madhya Pradesh Tiger Foundation Society, Bhopal for the financial support. National Park and Zoo authorities for the smooth sampling and zoo keepers for their support. Last but not the least all the hands came forward to make this investigation and manuscript happened. 

The experiment was conducted as per the guidelines of Institutional Animal Ethics Committee (IAEC) vide order no. 56/IAEC/Vety. /2020 dated 27/10/2021 and Principal Chief Conservator Forest (PCCF), M.P. approval no. Draft men-II/Research/2890 dated 30.03.2021. 

The authors declare that they have no conflict of interest.