MINISTRY OF EDUCATION

AND TRAINING VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

-----------------------------

Nguyen Thi Thu Hien

BIOLOGICAL CHARACTERISTICS AND

EFFECTS OF GONADOTROPIN ON

REPRODUCTIVE ABILITY OF COMMON PALM CIVETS

(Paradoxurus hermaphroditus Pallas, 1777) IN CAPTIVITY

Major: Biotechnology

Code: 9 42 02 01

SUMMARY OF BIOLOGY DOCTORAL THESIS

Ho Chi Minh City –2019

The thesis was completed at: Graduate University of Science and

Technology - Vietnam Academy of Science and Technology.

Supervisor 1: Assoc. Prof. Dr. Nguyen Thanh Binh

Supervisor 2: Assoc. Prof. Dr. Nguyen Thi Phuong Thao

Reviewer 1: …

Reviewer 2: …

Reviewer 3: ….

The thesis will be defended at The Thesis Evaluation Council held at Graduate University of Science and Technology - Vietnam

Academy of Science and Technology at [time][date] [month] [2019].

Thesis can be found at: - The Library of Graduate University of Science and Technology -

Vietnam Academy of Science and Technology

- The National Library of Vietnam

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Introduction

1. The urgency of the thesis The Common Palm Civet (Paradoxurus hermaphroditus Pallas, 1777) belongs to the Viverridae family, carnivora (Carnivora). This animal is widely distributed in Central, South and Southeast Asia. In Vietnam, the civets are widely distributed across the country [7]. Hunting and use of civet taps for different purposes such as meat, leather, fur, aromatherapy, used in the production of weasel coffee along with their declining habitat are depleting this species in nature [2, 8]. Preserving and storing genetic resources is one of the urgent, regular and long-term solutions [9]. In order to sustainably preserve livestock gene sources, the exploitation and development of genetic resources is an effective solution [10]. Therefore, in Vietnam, successfully built many breeding civet farms. The civet farming in addition to bringing high economic efficiency to the farmers also helps to reduce hunting and contribute to preserving biodiversity [11]. In breeding management and improvement of the civet process, studying their biological characteristics and increasing their reproductive performance are very important. Therefore, the topic "Biological characteristics and effects of gonadotropin on reproductive ability of common palm civets (Paradoxurus hermaphroditus Pallas, 1777) in captivity" is carried out.

2. The aims of the thesis - Identify biological characteristics, some indicators of physiological and biochemical blood and urine of civets in captivity.

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- Determine the change in sex hormones of female civets and evaluate the effect of sexual stimulation on the fertility of civets in captivity in order to preserve herd in the direction of quantity development. 3. The main contents of the thesis - Study on biological characteristics (appearance, behavior, growth, reproduction) of the civets in captivity - Studying on physiological and biochemical parameters of the civets in captivity. - Study on the change of sex hormones of the female civets in captivity. - Study on the effect of gonadotropin (PMSG, HCG) on the reproduction of female civets.

CHAPTER 1. LITERATURE OVERVIEW 1.1. Overview of the common palm civets This section presents an overview of the characteristics of species, appearance, behavior, reproduction, distribution of species in nature and current situation of breeding civets. 1.2. Overview of research on the common palm civets This section presents research on the civets in the world and in Vietnam. The studies mainly focused on the behavior, physiological and biochemical characteristics of this species in nature, in terms of genetic diversity and phylogeny. 1.3. Overview of PMSG and HCG An overview of structure, functions, applications and studying of the use of PMSG and HCG in livestock. 1.4. Overview of estrogen and progesterone

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An overview of the structure and function of estrogen and progesterone; method of extracting and identifying them through faeces.

CHAPTER 2. MATERIALS AND METHODS

2.1. Location, time of study 2.1.1. Location

- Biotechnology Application Center (Biotechnology) in Xuan Duong commune, Cam My district, Dong Nai province.

- Thanh Long Wildlife Farm, Thu Duc District, Ho Chi Minh City.

- Experimental Research Center Thu Dau Mot University.

- Center for Animal Biotechnology - Southern Animal Husbandry Institute.

2.1.2. Time: from May 2016 to March 2019. 2.2. Farms, food, water 2.3. Contents and methods 2.3.1. Study some biological characteristics of the civets in captivity

2.3.1.1. Material Growth characteristics: Select 64 individuals (32 males, 32

females) to monitor the growth indicators continuously from 3 to 24 months of age.

Reproductive characteristics: Conducted follow-up on 32 female civets and 34 male civets before sexual maturity; 42 mature female civets

2.3.1.2. Survey targets - Physical characteristics, some adaptive behavior of the civets in

captivity: activity, nutritional characteristics.

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- Growth indicators of the civets in captivity: weight, length body, length tail, chest girth.

- Some breeding criteria of female civets and male civets in captivity.

2.3.1.3. Methods of determining the dimensions and dimensions of measuring dimensions

Measure dimensions: body length (HB), tail length (T), chest girth (C) and weight (W) according to Dang Huy Huynh et al. (2010).

2.3.1.4. Methods of growth rates Absolute growth (A): A = V2– V1 / t2 - t1 Relative growth (R%): R (%) = 2 (V2– V1) x 100 / (V1 + V2). In

which: V1, V2 is the volume value (size) of the survey at t1, t2. 2.3.1.5. Methods of studying reproductive characteristics -Experimental method: direct monitoring and attaching

surveillance cameras to breeding facilities. Parameters were collected and recorded for each experimental individual.

-Methods of interview: Interview farmers directly about some reproductive characteristics of the civets. 2.3.2. Researching some physiological and biochemical parameters of the civets in captivity

2.3.2.1. Materials A total of 186 blood samples were collected from 62 civets (30 males, 32 females). The civets were not sick, the females were not pregnant.

2.3.2.2. Survey targets Survey of 18 blood physiological indicators, 14 blood and urine biochemical parameters. All indicators were monitored in normal health status, by age and by gender.

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2.3.2.3. Blood sampling and analysis - Blood samples were collected through the tail peduncle vein with

3ml syringe (needle size 25Gx1). - Blood physiological indicators were performed by using

automated hematology analyzer Mindray BC 2800 Vet. - Blood biochemical parameters were measured by chemical

analyzer (Abaxis Vetscan 2, Union City, CA, USA). 2.3.2.4. Urinary sampling and analysis - Urinary samples were collected from the stainless steel floor of a

special barn by cylinder, 18-20 hours, 1 time/week for 1 month (for each individual).

- Urinary biochemical norms were measured on an automated analyzer (model Teco TC-101, Teco diagnostics, USA).

- Na+, K+ and Cl- analyzes were measured from the supernatant obtained by the centrifugation of urine samples from the civets at 3000 rpm for 10 min (Roto x 32®-Hettich) and were performed on an ion selective device (model Roche 9180, Roche Diagnostics, Switzerland). 2.3.3. Study on changes in sex hormone content of female civets in captivity

2.3.3.1. Materials Fecal samples were collected from 12 mature female civets for 16

months. 2.3.3.2. Survey targets Changes in the endocrine index: estradiol (E2), progesterone (P4)

of adult female civets in the following cases: non-pregnant, pregnant and pseudopregnancy.

2.3.3.3. Fecal sampling and extraction

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Fecal samples were collected an average of 3 days, about 18h - 20h in 14 months. Fresh samples (5g) were collected in a plastic bag and stored (-200C) until analysis. After thawing, 0.2 g was weighed and placed in a glass jar containing 2 ml of 90% methanol. After shaking for 30 min, the suspension was centrifuged at 1,700 x g for 20 min (on a shaker EAB 20). After centrifugation, approximately 1 ml of the aqueous solution was extracted into 1.5 ml eppendorf vial and frozen (-200C) until using (Frederick et al., 2010). The remainder is returned to the glass vial and dried to determine the dry weight of the stool.

2.3.3.4. Hormone assays The fecal contents of P4 and E2 were determinned by using

fully automatic ELISA Dynex DS2 (Dynex, USA), Progesterone and Estradiol ELISA Kit.

2.3.4. Study the effect of gonadotropin (PMSG, HCG) on reproductive ability of the civets

2.3.4.1. Materials A total of 54 mature female civets, after the survey were

classified into 3 groups: - Group 1: Civets were slow to breed for the first time (after 24

months of age, there was no estrus manifestation); n = 14. - Group 2: Slowly rejuvenating civets (after 12 months after birth,

no estrus have been seen); n = 15. - Group 3: Low productivity breeding civets (1 litter/year;

number of civets per litter was small, 1-2 newborn/litter); n = 25. 2.3.4.2. Survey targets - Changes in endocrine indexes: estradiol (E2), progesterone (P4)

after injection of PMSG and HCG (from 2 days before injection (day

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-2) to the injection date (day 0) and 8 days later treatment. - The duration of the civets’ oestrus after injection of PMSG and

HCG. - Ratio of estrus civets (estrus civets/total number of treated

civets). - Percentage of pregnancy civets (number of pregnant civets/total

number of treated civets). - The average of newborn civets on the litter. - Average weight of newborn civets. - The proportion of live civets after 24 hours and after 1 month

(number of surviving civets /total number of newborn civets). 2.3.4.3 Type of reproductive hormones Mixed PMSG / HCG (ratio 2: 1): trade name is Gestavet (United

Kingdom). Each vial contained 400 IU PMSG/200 IU HCG and vial containing 5ml solvent for injection solution.

2.3.4.4. The treatments for injecting reproductive hormones Control (ĐC): 0 IU PMSG + 0 IU HCG (No injection) Treatment 1 (CT1): 20 IU PMSG + 10 IU HCG Treatment 2 (CT2): 30 IU PMSG + 15 IU HCG Treatment 3 (CT3): 40 IU PMSG + 20 IU HCG The experiment was performed all treatments for civets in 3

groups. Each batch has 3 or more civets. 2.3.4.5. Experimental layouts Table 2. 2. Experimental layout diagram

Group ĐC CT1 CT2 CT3

Group 1 (n=14) 3 4 4 3

Group 2 (n=15) 3 4 4 4

Group 3 (n=25) 3 7 7 8

2.3.4.6. Injection process

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The female civets are injected intramuscularly (IM) at 8 AM. 2.3.4.7. Methods of determining hormone changes Sampling procedure, stool extract, hormone test is done similarly

2.3.3.3 and 2.3.3.4. 2.3.4.8. Method of determining oestrus - Observation method. - Clinical method: monitoring pregnancy and childbirth of female

civets. 2.4. Data analysis

From the data obtained, conduct the calculation of statistical parameters: average value (X̅), standard deviation (SD); ANOVA analysis of a factor with a level of α = 0.05. Calculations were processed by MS-Excel 2013 software.

CHAPTER 3. RESULTS AND DISCUSSION 3.1. Results of studying biological characteristics 3.1.1. Morphological characteristics and some behaviors of

civets in culture conditions In terms of morphology, the civets in the captivity also has the

characteristics of the species as in natural conditions. The coat is gray or moldy, sucking black fur; Vertebral vertebrae, black-brown ribs or often forming three stripes along the spine from the shoulders to the base of the ears. The tail has unknown streaks or is black at the base of the tail, the tail is usually black, but in some civets it may be white; the nose, cheeks, ears, lower thighs and four legs are black; gray belly. When the civets are still young, the black brown stripe is not clear, the hair is rough. The more you grow, the smoother the fur and the

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dark brown the clearer. The face has 2-3 bright spots on the forehead or the edge of the eyes.

Regarding nutritional characteristics, the civets are omnivorous. Survey results showed that civets eaten many fruits. The favorite fruit of civets is bananas. In breeding, depending on the season, it is possible to change different food sources. In terms of animal feed, civets especially like to eat meat, fish and eggs. According to Dang Huy Huynh et al., (2010); Duckworth (2016), the civets is omnivorous, they can eat most of the food that humans eat [7, 111].

The civets mainly act at night, sleep at day. Excretion activities are mainly carried out at the beginning of the evening operation phase. Civets has very high protective behavior. When grazing another child into a barn, they will bite each other. The civet is only paired when the offspring has arousal. 3.1.2. Growth characteristics of the civet civet in captivity 3.1.2.1. Volume growth Survey results of mass growth rate of 64 civets were shown in Table 3.1. Table 3. 1. Growth rate of the civets’ weight in captivity

Table 3.1 showed that the amount of civets that was monitored

has an uneven growth rate over the ages of months, which was consistent with the periodic growth rules. Volume growth tended to increase gradually from 3-12 months of age, then gradually slow down

X̅(g)

Sx Cv% A (g/head/day)

Rw% X̅(g)

Sx Cv% A (g/head/day

Rw%

3 782 118.5 15.15 727 82.7 11.38 55 >0,056 1,152 126.1 10.95 4,11 a 38,26 a 975 125.3 12.85 2,76 b

29,14b 177 <0,05

9 1,735 109.1 6.29 6.48 40.39 1,456 117.8 8.09 5.34 39.57 279 <0,0512 2,644 113.5 4.29 10.10 41.52 2,225 113.7 5.11 8.54 41.78 419 <0,0515 3,245 128.1 3.95 6.68 20.41 2,848 120.7 4.24 6.92 24.56 397 <0,0518 3,533 108.5 3.07 3.20 8.50 3,175 118.9 3.74 3.63 10.86 358 <0,0521 3,743 116.3 3.11 2.33 5.77 3,335 84.7 2.54 1.78 4.92 408 <0,0524 3,925 105.2 2.68 2.02 4.75 3,516 93 2.63 2.01 5.28 409 <0,05X̅ 5.14 20.22 4.71 21.16

Age (month)

Male (n=32) (1) Female (n=32) (2)X̅1 - X̅2 P

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from 12-24 months. The period with the highest growth rate was from 9 to 12 months with the average absolute growth of each male being 10.1 g/head/day, growing relatively R% = 41.52%, These indicators corresponded to females of 8.54 g/head/day and R% of 41.78%.

3.1.2.2. Growth in body length The survey results of growth of civets’ body length were shown in Table 3.2. Table 3 2. Growth rate of body length of the civets in captivity

Table 3.2 showed that the growth rate of the civets’ body

length was slow down through the months of age. The fastest growth phase of body length was the period of 3-6 months with absolute growth of 2.87 cm/head/month, relative growth R% = 19.46% (for the period of 3 months) in males and 2.57 cm/head/month, relative growth of R% = 18.15% in females. After the 15th month, when the stem length was close to the good limit of the species, the growth in the period of 18-24 months was very small (0.10-0.76 cm/head/month).

3.1.2.3. Growth in tail length The survey results of the civets’ tail length growth were shown in Table 3.3. Table 3. 3. Growth rate of the civets’ tail length in captivity

X̅(g)

Sx Cv% A (cm/head/m)

Rw% X̅(g)

Sx Cv% A (cm/head/m)

Rw%

3 39.89 0.38 0.95 38.67 0.32 0.83 1.22 >0,056 48.49 0.44 0.91 2.87 19.46 46.39 0.46 0.99 2.57 18.15 2.10 >0,059 56.27 0.46 0.82 2.59 14.85 53.24 0.53 1.00 2.28 13.75 3.03 <0,05

12 62.55 0.55 0.88 2.09 10.57 59.01 0.55 0.93 1.92 10.28 3.54 <0,0515 68.92 0.45 0.65 2.12 9.69 65.12 0.54 0.83 2.04 9.84 3.80 <0,0518 70.36 0.77 1.09 0.48 2.07 67.41 0.31 0.46 0.76 3.46 2.95 <0,0521 71.33 0.41 0.57 0.32 1.37 68.68 0.29 0.42 0.42 1.87 2.65 <0,0524 71.62 0.41 0.57 0.10 0.41 69.03 0.30 0.43 0.12 0.51 2.59 <0,05X ̅ 1.51 8.35 1.45 8.27

Age (month)

Male (n=32) (1) Female (n=32) (2)X̅1 - X̅2 P

X̅(g) Sx Cv%

A (cm/head/m) Rw%

X̅(g) Sx Cv%

A (cm/head/m) Rw%

3 36.44 0.93 2.55 36.02 1.18 3.28 0.42 >0,056 38.24 0.95 2.48 0.60 4.82 37.68 1.28 3.40 0.55 4.50 0.56 >0,059 41.45 0.91 2.20 1.07 8.06 40.82 1.18 2.89 1.05 8.00 0.63 >0,05

12 45.36 0.91 2.01 1.30 9.01 44.68 1.33 2.98 1.29 9.03 0.68 >0,0515 48.64 0.91 1.87 1.09 6.98 48.19 1.34 2.78 1.17 7.56 0.45 >0,0518 51.37 0.87 1.69 0.91 5.46 50.88 1.22 2.40 0.90 5.43 0.49 >0,0521 54.25 0.80 1.47 0.96 5.45 53.57 1.25 2.33 0.90 5.15 0.68 >0,0524 56.31 0.78 1.39 0.69 3.73 55.62 1.03 1.85 0.68 3.75 0.69 >0,05X ̅ 0.95 6.21 0.93 6.20

Age (month)

Male (n=32) (1) Female (n=32) (2)X̅1 - X̅2 P

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Table 3.3 shows that the growth of the tail length of the civets increased relatively evenly over the period of age, however, increased faster in the period of 6-12 months (R% from 8-10, 14%). The absolute growth rate (A) averageed 0.95 cm/head/month, the relative growth rate (R%) was 6.21% (in males) and A = 0.93 cm/head/month, R% = 6.20% (in females).

3.1.2.4. Chest growth The results of monitoring the dimension of chest girth of the

civets in captivity were shown in Table 3.4. Table 3. 4. Growth rate of the civets’ chest girth in captivity

Table 3.4 showed that the chest girth growth rate was high in the

period of 3-12 months of age and highest in the 9-month period (A = 1.27 cm/head/month, R = 15.07% in males) and A = 1.06 cm/ head/month, R = 13.21% in females). This was in accordance with the rules of fast chest growth rate in the sexual maturity stage in animals. Age of sexual maturity of civets was in the period of 9-12 months of age [111]. 3.1.3. Reproductive characteristics of incense civet in captivity

3.1.3.1. Age of sexual maturity and estrus manifestations Results of monitoring the age of sexual maturity of civets (32

female civets, 34 male civets) in captivity in the study were shown in Table 3.5.

X̅(g) Sx Cv%

A (cm/head/m) Rw%

X̅(g)Sx Cv%

A (cm/head/m) Rw%

3 21.21 0.92 4.34 20.83 0.81 3.89 0.38 >0,056 23.43 0.83 3.54 0.74 9.95 22.56 0.71 3.15 0.58 7.97 0.87 >0,059 27.25 0.75 2.75 1.27 15.07 25.75 0.70 2.72 1.06 13.21 1.50 <0,05

12 29.36 0.93 3.17 0.70 7.45 27.64 0.70 2.53 0.63 7.08 1.72 <0,0515 29.87 0.92 3.08 0.17 1.72 28.24 0.74 2.62 0.20 2.15 1.63 <0,0518 30.32 0.90 2.97 0.15 1.50 28.68 0.66 2.30 0.15 1.55 1.64 <0,0521 30.54 0.86 2.82 0.07 0.72 28.93 0.82 2.83 0.08 0.87 1.61 <0,0524 30.66 0.85 2.77 0.04 0.39 29.12 0.78 2.68 0.06 0.65 1.54 <0,05X ̅ 0.45 5.26 0.39 4.78

Age (month)

Male (n=32) (1) Female (n=32) (2)

X̅1 - X̅2 P

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Table 3. 5. Age of sexual maturity of the civets in captivity

Note: Different characters in the same row are statistically

significant differences (P <0.05). The table 3.5 showed that the female civets were starting to show

oestrus in the period of 10-14 months with an average weight of 2.38 - 2.62 kg. The average maturity was 11.96 months, with an average weight of 2.50 kg. According to Nelson (2013), the estrus of civets was between 11 and 12 months of age [112].

3.1.2.2. Mating activity, pregnancy rate and pregnancy time During the study period, we monitored the reproductive results of

42 female civets, with 84 pairings. Results of monitoring pregnancy rate and gestation period were presented in Table 3.6. Table 3. 6. Pregnancy ratio and duration in the civets

Note: the characters in the same column are different, the difference is statistically significant (P <0.05).

Age (month) Number

Rate(%) Weight (kg)

Age (month) Number

Rate(%) Weight

(kg)

9 0 0 9 2 5.88 1.8410 4 12.50 2.38 10 9 26.47 2.4511 5 15.63 2.43 11 14 41.18 2.5612 9 28.13 2.51 12 6 17.65 2.6113 6 18.75 2.58 13 3 8.82 2.8614 3 9.38 2.62 14 0 0.00

Not mature 24-30 5 15.63 3.26 0 0X̅ 11,96a 2,50 10,97b 2,52SD 1.22 0.08 1.03 0.04

Parameter

Female (n=32) Male (n=34)

Mature

PlaceNo of mating civets (n=84)

No of pregnancy

civets (heads)R (%)

Gestation length time (days)

Dong nai 30 14 46,67a 61,2

Thu Duc 54 42 77,78b 60,8X̅ 66.67 60.9SD 1.3

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3.1.3.3. Number of births per litter, survival rate, neonatal characteristics and weaning age

Results of monitoring the number of young civets on litter, neonatal characteristics and survival rates of 56 monitored parities were presented in Table 3.7. Table 3 .7. The number of piglets born on the litter and the survival rate

Note: the characters in the same column are different, the differences are statistically significant (P <0.05).

Table 3.7 showed that the births of each litter were range from 1 - 4. The average number of civets per litter of all 56 litters was 2.38. In nature, according to Dang Huy Huynh et al., (2010), civets lay 2 to 4 newborns [7]. According to Nelson (2013), 2-5 newborns, an average of 3.4 newborn civets /litter [112]. Newborn civets were very small and weak, unable to stand firmly, with an average weight of 95.16 grams. After a period of 7-10 days, the rim of the ear opened, from 12-15 days the eyes opened.

At birth 24h 48h 1 week 1 monthAt

Weaning Weight at birth

Weight at

weaning 1 42 63 34 1

X̅ 1 1,86a 96,93 585,36SD 0.77 7.45 26.85

1 62 153 144 7

X̅ 2 2,55b 94,57 59142SD 0.92 6.27 19.38X ̅ 2.38 96.15 90.03 87.22 81.53 80.83 80.83 95.16 590.83

Dong Nai (n= 14)

22 20 20 17

Place

Number of

newborn civets (con)

Number of (con) n=56

Live newborn civets(n / %)

Weight (X̅ , gr)

17 1796.93 585.36

84.62 76.92 76.92 65.38 65.38 65.38

Thu Duc (n=42)

107 101 97 93 9294.57 592.65

100.00 94.39 90.65 86.92 85.98 85.98

92

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3.2. Research results of some physiological and biochemical indicators of blood of civets in captivity 3.2.1. Hematological parameters of common palm civets by gender

The results of the study on the physiological parameters of 186 blood samples from 62 civets (30 males and 32 females) were presented in Table 3.8. Table 3.8.Hematological data of common palm civets by gender group

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3.2.2. Hematological parameters of common palm civets by age The results of the study on the physiological parameters of 186

blood samples from 62 civets by age were presented in Table 3.9. Table 3.9. Hematological data of common palm civets by age group

3.2.3. Serum biochemical parameters of common palm civets The results of blood biochemical parameters by sex and gender

group were shown in Table 3.10. and Table 3.11. Table 3.10. showed that the total serum protein (TP), globulin, albumin and phosphorus levels in males were higher than females, the difference was statistically significant (P <0.05).

16 Table 3.10. Serum biochemical parameter of commom palm civets by sex group

Table 3. 11. Serum biochemical parameter of commom palm civets by age group

17 3.2.4. Urinary biochemical parameters of common palm civets by sex

The results of the study on the urinary biochemical parameters from 60 civets were presented in Table 3.12.

Table 3.12. Urinary biochemical data of commom palm civets by sex group

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3.2.5. Urinary biochemical parameters of common palm civets by age

The results of urine biochemical indexes of civets in the age group were presented in Table 3.13. Table 3.13. Urinary biochemical data of commom palm civets by age group

Parameter 3-<12 months (n= 31) >12 months (n=31)

Mean ± SD Mean ± SD

Weight (g) 1.735±109,1a 3.335 ±84,7b

Body legth (mm) 56,27±0,46a 71,62±0,41b

Urobilinogen (µmol/L) 10,44 ±1,06 10,88 ±1,27

Glucose (mmol/L) Neg1a 0,47±0,22b

Billirubin (µmol/L) 0,46±0,06 0,35±0,05

Ketone (mmol/L) 0,13±0,02 0,17±0,03

Specific Gravity 1,02±0,01 1,02±0,01

Blood (Ery/µL) neg neg

pH 7,55±0,17 7,51±0,52

Protein (g/L) 16,01±1,27 15,88±1,31

Nitrite neg neg

Leukocytes (Leu/µL) 3,82 ± 0,12a 5,35 ± 0,37b

Ascorbic acid (mmol/L) 0,15±0,03 0,17±0,05

K (mmol/L) 173,23±43,12 185,27±51,25

Na (mmol/L) 69,86±11,07a 83,75±16,32b

Cl (mmol/L) 149,58±43,42 157,47±32,24

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3.4. The results on the changes of sex hormone of female civets in captivity

3.4.1. The changes of estradiol and progesterone in non-pregnant civets 3.4.1.1. Faecal estradiol in non-pregnant civets

The concentrations of fecal E2 in non-pregnant civets in our study ranged from 0.05 to 7.01 µg/g df, with an average of 1.07 ± 0.84 µg/g df and a peak of 3.22 ± 0.64 µg/g df. Although faecal estradiol value of civets have not been published, faecal estradiol values in other animals have already been widely used to monitor their sexual activities. For example, faeal E2 of the Siberian tiger population ranged from 0.39 to 0.49 µg/g and the mean faecal E2 of Bengal tiger was 0.45 µg/g, and thoat of Sumatra tiger was 2.36 µg/g [89].

Changes in E2 levels showed a cyclic fertilization. The duration of each cycle ranged from 26.8-33.1 days with the average of 28.6 ± 2.29 days. This period was comparable with that (27.0 days) of Siberian tigers [89] and of Bengal tigers (29.3 days) [88], but different from that of domestic cat (21 days) or leopard (10-20 days)[120]. 3.4.1.2. Faecal progesterone in non-pregnant civets In this study group, non-pregnant female civets’ fecal progesterone (P4) metabolites levels ranged from 0.15 to 12.32 µg/g df with the overall mean of 1.72 ± 2.16 µg/g df . For comparison, faecal progesterone level of Siberian tigers varies from 0.27 to 38.19 µg/g and that of the Sumatra tigers ranged from 0.09 to 18.52 µg/g, and the level in Bengal Tigers was 36.05µg/g [89].

Faecal progesterone levels of civets also changed over time. The peak of faecal progesterone level ranged from 6.03-12.32 µg/g df with an average of 7.26 ± 1.11 µg/g df. The cycle of change in progesterone level ranged from 26.6 to 31.0 days with an average of 27.8 ± 2.80 days.

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3.4.2. The changes of estradiol and progesterone in pregnant civets During pregnancy, the civets’ faecal P4 level ranged from 6.21

to 23.12 µg/g df with an average of 15.17 ± 5.22 µg/g df. This value was approximately 5 to 7 times higher (P <0.05) than non-pregnant and post-fertilization periods. In individuals with conception, P4 increased significantly for a period between 60 and 63 days after fertilization. Faecal E2 concentration of a juvenile fennel during pregnancy was relatively lower than that in other periods. Fecal E2 ranged from 0.22 to 1.05 µg/g df with an average of 0.74 ± 0.23 µg/g df. After parturition, E2 increased and marked the recovery of ovarian activity from 25-30 days. In contrast, in fetal predisposed individuals, there was a negligible E2 change (range of 0.35-1.99 µg/g df) compared to non-pregnant ones (P>0.05), and distinctly lower than that of the pregnancy period (P <0.05). This result was similar to that observed in the Pallas' cat, the clouded leopard, Tiger, in these an increase in estrogen excretion in faeces was not observed during pregnancy [120].

3.4.3. The changes of estradiol and progesterone in pseudopregnancy civets

In pseudopregnancy civets, there were also significant changes in P4 after fertilization, but the change duration was only 26-30 days. The faecal P4 level at this stage ranged from 8.02 to 11.47 µg/g df with an average of 9.73 ± 1.73 µg/g df. This value was significantly higher than that of non-pregnant civets but was significantly lower than those of pregnant animals (P<0.05). In other studies, leopard cats, clouded leopards, snow leopards and cheetahs have been reported to have increased duration of P4 contents during presumed pseudopregnancy [89]. Thus, the main indicator to distinguish between pregnancy and fake pregnancies is both the duration and the degree of the increase in the fecal P4.

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3.5. Results of studying the effects of sex hormones (PMSG, HCG) on fertility of the civets

3.5.1. The changes of estradiol and progesterone after injecting sex hormones

Results of monitoring E2 and P4 changes showed that E2 content in the feces of the civets in the treatments began to increase at day 1 after injection, peaking on day 2 and then gradually decreasing from day to day. 3rd, the average E2 content of day 2 when peaking in formula 1 was 2.59 µg/g df, treatment 2 was 2.69 µg / g df and treatment 3 was 3.16 µg/g df ; in which the highest was in treatment 3. Thus, when the higher treatment dose (40IU PMSG) increased the amount of sex hormones in the treated civet group (P <0.05). Meanwhile in the control group, E2 content in the stool changed over the days, but this change was not significant (P> 0.05). By the 8th day, the hormone content in the stool in all treatments decreased to close to the value before sexual stimulation (day -2 and day -1).

3.5.2. Time appears manifestations and prolonged estrus In all experimental groups, the average duration of oestrus was

1.1 days (CT3 in group 2) to 2.6 days (CT1 in group 2 and group 3) after injection of sex hormones. In particular, CT3 always has the earliest time of estrus (from 1-1.5 days). The duration of oestrus was strongly correlated with the peak time of E2 (R = 0.82). The duration of oestrus in all formulas ranged from 2.9 to 3.9 days, of which the shortest time was in CT1 and the longest was in CT3. This time was longer than the time of expression of oestrus in the civet without sexual stimulation during the breeding season (2-3 days). ANOVA analysis showed that there was a statistical difference (P <0.05) on the duration of oestrus and duration of oestrus between the formulas in each experimental civet group. CT3 always has the earliest occurrence of oestrus and the longest period of oestrus.

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3.5.3. The results of using hormone formulas on some reproductive indicators

Results of monitoring reproductive efficiency after injection of sex hormones were presented in table 3.19. Table 3. 19. Results of reproductive performance monitoring after injecting sex

hormones Treatment ĐC CT1 CT 2 CT 3

n 9 15 14 15

Estrus civets Civets 1 12 12 14 % 11,11a 80,00b 85,71c 93,33d

Pregnant civets Civets 1 10 10 12 % 11,11a 66,67b 71,43c 86,67d

Newborn/ litter (X̅ ± SD) Civets/litter 2±0a

3,04± 0,47b

3,41± 0,66c

3,53± 0,90c

Weight at birth (X̅ ± SD) (g) 95,5±0

96,15± 2,14

95,72± 2,17

94,02± 3,35

Survivall rate after 48h (%) 100,00a

86,60b

86,35b

90,00b

Survivall rate after 1 month (%) 100,00a

83,57b

82,57b

82,83b

Note: The difference of the characters in the same row is statistically different (P <0.05), according to the T-test test with the significance level α = 0, 05.

CONCLUTIONS AND RECOMMENDATIONS 1. Conclusion 1.1. Biological characteristics of the civets in captivity

- Description of morphological characteristics, nutritional characteristics, weight growth, body length, tail length and chest girth of the civets in the period of 3-24 months.

- Identified some reproductive characteristics of the civets: The female civets have an average sexual maturity of 11.96 months and in the male civets is 10.97 months. The average gestation period is 60.9 days. The number of civets born in each litter ranges from 1-4 heads,

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averaging of 2.38 civets/litter. Newborn civet weighs have an average of 95.16 grams. 1.2. Physiological - biochemical blood and urine indicators of the civets

- Identified 18 physiological indicators and 14 blood biochemical indicators in the civets by age and gender group. The average number of erythrocytes is 11.06 x 1012/L, hemoglobin content of 120.74 gr/L. The average number of white blood cells is 12.32 x 109/L; Leukocyte formula: Lymphocyte 44.24%, Monocyte 3.96%, Gran 51.8%. The average platelet count is 336.25 x 109/L.

- Identified 14 urine biochemical indicators of the civets: leukocyte (4.63 Leu/µL), nitrite (negative), erythrocyte (negative), Protein (15.94 g/L), Glucose (0.46 mmol/L), Keton body (0.15 mmol/L), Bilirubin (0.41 µmol/L); average density (SG) is 1.02, pH is 7.53; Urobilinogen (UBG) is 0.1 µmol/L; K+ is 179.25 mmol/L, Cl- is 153.53 mmol/L, Na+ is 76.82 mmol / L. 1.3. The changing sex hormones of the female civets in captivity

- Identified changes in hormones estradiol (E2) and progesterone (P4) in the feces of the civets at the stage of non-pregnancy, pregnancy and pseudopregnancy.

- During pregnancy, the P4 level in the civets is 5 to 7 times higher than that of the non-pregnant period. P4 can be used as an indicator of determining pregnancy in the civets.

1.4. The effects of sex hormones (PMSG, HCG) on the reproduction ability of female civets.

- Determination of the effect of gonadotropin on hormone changes E2 and P4 after injection and oestrus time after injection of sex hormones.

- Determined the effect of PMSG and HCG on the reproductive efficiency of the civets. Percentage of oestrus and pregnancy increases; The number of newborn civets on litter was significantly higher than the control. The dose of 40 IU PMSG/ 20IU HCG injection gives the highest reproductive efficiency. Sex hormones do not affect neonatal weight and survival. 2. Recommendations

- Applying the results of studying physiological, biochemical and blood parameters on the civets in clinical examination, disease diagnosis and care of this species in culture conditions. Need to

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continue to study the biochemical indicators of blood and urine of the civets in pathological cases.

- In the civets, analyzing hormone in stool is a useful method to confirm the period of oestrus, pregnancy, non-pregnancy or false pregnancy.

- The use of gonadotropin to bring about positive results, the 40 IU PMSG / 20 IU HCG injection dose gives the highest effect. The use of ovarian ultrasound and observation of vaginal cells should be combined to contribute to the techniques of assisting reproduction of this species in culture conditions.

NEW CONTRIBUTIONS OF THE THESIS 1. The thesis is the first scientific work to systematically study

the biological characteristics (appearance, nutrition, behaviors, growth, reproduction) of the common palm civets in captivity in Vietnam. The thesis provided important data contributing to the improvement of breeding techniques; increase the effectiveness of civet conservation; paving the way for similar studies on other wildlife species in captivity.

2. Physiological and biochemical indicators of blood, urine by age group and sex group of the common palm civets published in Vietnam for the first time. These results are the scientific basis for research, diagnosis and wildlife breeding.

3. This is the first work investigaved the change of reproductive endocrine indicators by non-invasive method in order to determine the estrus period of the common palm civets. The thesis has identified the changes of the hormone estradiol and progesterone in the faeces of the common palm civets in the period of non-pregnancy, pregnancy and presumed-pseudopregnancy.

4. The thesis evaluated the effects of gonadotropin (PMSG, HCG) on the change of estradiol and progesterone, on the reproductive ability of female civets in captivity. These results contributed the applications of assisted reproductive technologies in wildlife; deploy for farmers and wild animal breeding areas in order to improve reproductive efficiency for both exploit and preserve the ex-situ conservation of this rare animal.

SCIENTIFIC PUBLICATIONS

1. Nguyen Thi Thu Hien, Nguyen Thi Phuong Thao, Nguyen Thanh Binh, Growth characteristics of Common Palm Civets (Paradoxurus hermaphroditus Pallas, 1777) in captivity, VNU Journal of Science: Natural Science and Technology, 2017, 33 (1S): 207-213.

2. Nguyen Thi Thu Hien, Nguyen Thi Phuong Thao, Nguyen Thanh Binh, Study on hematological parameters of common palm civets (Paradoxurus hermaphroditus Pallas, 1777) in captivity. Journal of biotechnology, 2017, 15(3A): 71-76.

4. Nguyen Thi Thu Hien, Nguyen Thi Yen Nhi, Nguyen Thi Thanh Thao, Nguyen Thanh Binh, Effects of diets on producibility of raw weasel coffee of civets in captivity, Scientific journal of Thu Dau Mot University, 2017, 2 (33), 161-169.

4. Nguyen Thi Thu Hien, Nguyen Thi Phuong Thao, Nguyen Thanh Binh, Reproductive characteristics of civets (Paradoxurus hermaphroditus Pallas, 1777) in captivity, Proceeding of the 7Th national scientific conference on ecology and biological resources in Vietnam. Ha Noi, 2017, 694-701.

5. Nguyen Thi Thu Hien, Nguyen Thi Phuong Thao, Nguyen Thanh Binh, Effects of rations on producbility of raw weasel coffee of civets (Paradoxurus hermaphroditus Pallas, 1777) under captive condiction, Proceeding of national conference on animal and veterinary sciences, Can Tho, 2017, 283-289

6. Nguyen Thi Thu Hien, Nguyen Thi Phuong Thao, Nguyen Thanh Binh, A non-invasive technique to monitor reproductive hormone levels in common palm civets, Paradoxurus hermaphroditus Pallas, 1777. Academia Journal of Biology, 2018, 40(3): 74–81. https://doi.org/10.15625/2615-9023/v40n3.12654.

7. Nguyen Thi Thu Hien, Nguyen Thi Phuong Thao and Nguyen Thanh Binh, Blood and urinary biochemical parameters of the Commom Palm Civets (Paradoxurus hermaphroditus, Pallas 1777) in captivity, Journal of Animal Husbandry Sciences and Technics, 2018, 235, 90-96.