Summary of Biology doctoral thesis: Biological characteristics and effects of gonadotropin on reproductive ability of common palm civets (Paradoxurus hermaphroditus Pallas, 1777) in captivity

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Identify biological characteristics, some indicators of physiological and biochemical blood and urine of civets in captivity; 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.

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Nội dung Text: Summary of Biology doctoral thesis: Biological characteristics and effects of gonadotropin on reproductive ability of common palm civets (Paradoxurus hermaphroditus Pallas, 1777) in captivity

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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
1 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.
2 - 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
3 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.
4 - 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.
5 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
6 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
7 -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
8 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
9 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 Male (n=32) (1) Female (n=32) (2) Age A A X̅ 1 - X̅ 2 P (month) Sx Cv% Rw% Sx Cv% Rw% X̅ (g) (g/head/day) X̅ (g) (g/head/day 3 782 118.5 15.15 727 82.7 11.38 29,14 55 >0,05 a a b b 6 1,152 126.1 10.95 4,11 38,26 975 125.3 12.85 2,76 177
10 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 Male (n=32) (1) Female (n=32) (2) Age A A X̅ 1 - X̅ 2 P (month) Sx Cv% Rw% Sx Cv% Rw% X̅ (g) (cm/head/m) X̅ (g) (cm/head/m) 3 39.89 0.38 0.95 38.67 0.32 0.83 1.22 >0,05 6 48.49 0.44 0.91 2.87 19.46 46.39 0.46 0.99 2.57 18.15 2.10 >0,05 9 56.27 0.46 0.82 2.59 14.85 53.24 0.53 1.00 2.28 13.75 3.03 0,05 21 54.25 0.80 1.47 0.96 5.45 53.57 1.25 2.33 0.90 5.15 0.68 >0,05 24 56.31 0.78 1.39 0.69 3.73 55.62 1.03 1.85 0.68 3.75 0.69 >0,05 X̅ 0.95 6.21 0.93 6.20
11 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 Male (n=32) (1) Female (n=32) (2) Age X̅ (g) A A X̅ 1 - X̅ 2 P (month) X̅ (g) Sx Cv% Rw% Sx Cv% Rw% (cm/head/m) (cm/head/m) 3 21.21 0.92 4.34 20.83 0.81 3.89 0.38 >0,05 6 23.43 0.83 3.54 0.74 9.95 22.56 0.71 3.15 0.58 7.97 0.87 >0,05 9 27.25 0.75 2.75 1.27 15.07 25.75 0.70 2.72 1.06 13.21 1.50
12 Table 3. 5. Age of sexual maturity of the civets in captivity Female (n=32) Male (n=34) Age Rate Age Rate Parameter Number Number Weight (month) (%) Weight (kg) (month) (%) (kg) 9 0 0 9 2 5.88 1.84 Mature 10 4 12.50 2.38 10 9 26.47 2.45 11 5 15.63 2.43 11 14 41.18 2.56 12 9 28.13 2.51 12 6 17.65 2.61 13 6 18.75 2.58 13 3 8.82 2.86 14 3 9.38 2.62 14 0 0.00 Not mature 24-30 5 15.63 3.26 0 0 ̅ X 11,96 a 2,50 10,97 b 2,52 SD 1.22 0.08 1.03 0.04 Note: Different characters in the same row are statistically significant differences (P
13 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 Number Live newborn civets Weight (X ̅ , gr) of (n / %) Number of Place newborn Weight (con) n=56 At Weight civets At birth 24h 48h 1 week 1 month at Weaning at birth (con) weaning 1 4 22 20 20 17 17 17 Dong Nai 2 6 96.93 585.36 (n= 14) 3 3 84.62 76.92 76.92 65.38 65.38 65.38 4 1 ̅X1 1,86 a 96,93 585,36 SD 0.77 7.45 26.85 1 6 107 101 97 93 92 92 Thu Duc 2 15 94.57 592.65 (n=42) 3 14 4 7 100.00 94.39 90.65 86.92 85.98 85.98 ̅X2 2,55 b 94,57 59142 SD 0.92 6.27 19.38 X̅ 2.38 96.15 90.03 87.22 81.53 80.83 80.83 95.16 590.83 Note: the characters in the same column are different, the differences are statistically significant (P
14 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
15 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
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
18 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 3-12 months (n=31) Parameter 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 1a Glucose (mmol/L) Neg 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 a Leukocytes (Leu/µL) 3,82 ± 0,12 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 a Na (mmol/L) 69,86±11,07 83,75±16,32b Cl (mmol/L) 149,58±43,42 157,47±32,24