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ISSN 2408-9346

Том 1. №2(2),

2014

Учредитель: ФГАОУ ВПО «Белгородский государственный национальный исследовательский университет»

ISSN 2408-9346

Volume 2(2), 2014 Founded by: Belgorod State University

ГЛАВНЫЙ РЕДАКТОР СЕРИИ: Присный А.А., кандидат биологических наук, доцент

EDITOR-IN-CHIEF OF A SERIES: Andrey A. Prisny, Ph.D. in Biology, Associate Professor

заместитель главного редактора СЕРИИ: Скоркина М.Ю., кандидат биологических наук, доцент

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РЕДАКТОР АНГЛИЙСКИХ ТЕКСТОВ СЕРИИ: Ляшенко И.В., кандидат филологических наук, доцент ЧЛЕНЫ РЕДАКЦИОННОЙ КОЛЛЕГИИ: Зотин А.А., доктор биологических наук, ведущий научный сотрудник Илюха В.А., доктор биологических наук Сафонова Т.А., доктор биологических наук, профессор Шапошников А.А., доктор биологических наук, профессор РЕДАКЦИОННЫЙ СОВЕТ: Кэкули-Дуарте, PhD, Ирландия Липунова Е.А., доктор биологических наук, профессор, Россия Литвинов Ю.Н., кандидат биологических наук, доцент, Россия Маслова М.Н., доктор биологических наук, профессор, Россия Чернявских С.Д., кандидат биологических наук, доцент, Россия

ENGLISH TEXT EDITOR: Igor V. Lyashenko, Ph.D. in Philology, Associate Professor EDITORIAL BOARD: Alexey A. Zotin, Doctor of Biology Viktor A. Ilukha, Doctor of Biology Tatyana A. Safonova, Doctor of Biology, Professor Andrey A. Shaposhnikov, Doctor of Biology, Professor EDITORIAL TEAM: Thomaé Kakouli-Duarte, Ph.D. in Biology, Lecturer in Biosciences, Ireland Elena A. Lipunova, Doctor of Biology, Professor, Russia Yuri N. Litninov, Ph.D. in Biology, Associate Professor, Russia Marina N. Maslova, Doctor of Biology, Professor, Russia Svetlana D. Chernyavskikh, Ph.D. in Biology, Associate Professor, Russia

СОДЕРЖАНИЕ

CONTENTS

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van Effect of time and temperature on the survival rate of mouse sperm (Mus musculus var. Albino) in short-term preservation without cryoprotectant agents....4

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong Examining the effects of lead on the life of larval zebrafish (1-7 days old).......................11

Hoa Pham Van, Quan Thai Ke The web - based tool for identification of amphibian and reptiles presented in three western provinces of South Eastern region, Vietnam...................................21

Hoa Pham Van, Quan Thai Ke The web - based tool for identification of amphibian and reptiles presented in three western provinces of South Eastern region, Vietnam.................................. 21

Чернявских С.Д., Голдаева К.А., Дрыганова Л.А., Филиппенко Е.Г. Фукциональные особенности сердечно-сосудистой системы у юношей призывного возраста...................33

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G. Functional peculi-arities of the cardiovascular system in young conscript males.............................................................. 33

Grebtsova E.A., Prisny A.A. Energetic and motion activity of hemocytes of Dictyoptera representatives.........................41

Grebtsova E.A., Prisny A.A. Energetic and motion activity of hemocytes of Dictyoptera representatives........................41

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СЕРИЯ

Физиология

А У Ч Н ЫЙ РЕЗУЛЬТАТ

УДК 576.3

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van

EFFECT OF TIME AND TEMPERATURE ON THE SURVIVAL RATE OF MOUSE SPERM (MUS MUSCULUS VAR. ALBINO) IN SHORT-TERM PRESERVATION WITHOUT CRYOPROTECTANT AGENTS

Abstract

n this study, we studied the use of physiological saline solution (NaCl 0,9%) or dulbecco’s phosphatebuffered saline (D-PBS) for mature sperms short-term preservation. After being collected from epididymides, sperms were adjusted to desired concentration (2x106 sperms/ml) with NaCl 0.9% solution or D-PBS solution (the dishes containing sperms were covered by mineral oil) and stored at 4oC, 10oC and room temperature (RT/26oC). The survival rate of sperms were evaluated by morphological observation and staining methods (single staining and double staining) every 8 hours. The results showed that the mouse sperms can be stored in a short-term at 4oC or 10oC without cryoprotectant agents, the survival rate in D-PBS solution are higher than those in NaCl 0,9% solution. The lethal concentration and time (LCt50) to mouse sperm in short-term preservation is 10 hours in D-PBS or NaCl 0,9% solution without cryoprotectant agents.

ey words: epididymis, mouse, short-term preservation, spermatozoa.

From the late twentieth century, the development of biology is very strong with many great achievements, especially in the studies about transgenation, cell culture, cloning, in vitro fertilization. They have contributed importantly in improving human’s life and preservation of genetic resources of many species. In particular, sperm preservation is the most effective method to preserve genetic resources of males which are valuable or in danger. The mammalian sperms have been preserved by freeze-drying or vitrification methods with variety levels of success; the survival rate after thawing is quite high. In general, these techniques require elaborate protocols using expensive apparatuses. Moreover, cryopreservation has disadvantages when used for shortterm storage, such as retaining the motility and fertility of the spermatozoa. In many cases, e.g. during the short interval between two experiments, or to be transferred from a farm to a lab, sperms need to be preserved only in a short time

[10]. The using the fresh semen short-term preservation would be particularly convenient since it does not require bacteriological monitoring, which is often laborious and time-consuming; and it does not require complicated method (spermatozoa freezing-thawing) [3, 9]; spermatozoa can be preserved without cryoprotectants and do not require a supply of liquid nitrogen as in the case of long-term storage and shipment [1]. Most of short-term sperm preservation methods mainly used commercial media (M2, KSOMAA, TYH medium) [9-11]. In mice, various methods of sperm storage without freezing have been tried, such as evaporative drying or storage in salt and sugars [7]. The mouse spermatozoa remains the fertilizing ability after being stored at 4-6oC in TYH medium in 7 days (Sankai et al. [8]); mouse sperm could be preserved at 4oC in KSOMAA medium with a high-salt concentration and osmolarity of 800 mOsm-l for 2 months (Van Thuan et al. [10]). The sperm preserved at

№2 2014

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van

EFFECT OF TIME AND TEMPERATURE ON THE SURVIVAL RATE OF MOUSE SPERM (MUS MUSCULUS VAR. ALBINO) IN SHORT-TERM PRESERVATION WITHOUT CRYOPROTECTANT AGENTS

4oC in medium with trehalose supplemented for 1 week or at RT in salt or sugar can be used as donor in ICSI [3, 7]. In this study, we were going to evaluate the use of two common solutions NaCl 0,9% and dulbecco’s phosphate-buffered saline (D-PBS) in short-term sperms preservation without cryoprotectant agents. Materials and methods

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in NaCl 0.9% or D-PBS solution were calculated every 8 hours. Experimental design Each experiment had 4 replicates. In each experiment, number of input sperms at initial time (0 hour) are the same. At each examined temperature, we evaluated the survival rate of sperm at the different time points: 0, 8, 16, 24, 32, 40 hours. Based on final result, the effects of solution, duration of preservation, and temperatures on the survival rate of mouse sperm in short-term preservation without cryoprotectant agents were determined. Consequently, an equation was set up to predict the survival rate of sperm under the influence of these factors. Statistical Analysis All data obtained from this study were calculated by Minitab 16, R software. Data are given as the mean ± SE. For all statistical tests, differences were considered statistically different at p < 0.05. Logistic regression analysis method with Poission regression model was used to analyze the correlation between the survival rate of sperms and examined factors. This model is   log  = α + β   a function: ; this means log of the survival rate of sperms is a function depend on x factor.

Animals Adult male mice (8 to 12 weeks old) were obtained from Laboratory of Stem cell Research and Application, University of Science, Ho Chi Minh city and Pasteur institute Ho Chi Minh city. All experimental animals were maintained in an room temperature condition and light controlled room (14:10-hours light-dark cycle with lights on at 7 a.m.). All experiment were carried out in the Laboratory of Anatomy-Human and Animal physiology, the University of education in Ho Chi Minh city. Sperm collection Epididymides collected from males using the euthanasia method would be placed directly into 1.5 ml eppendorfs containing NaCl 0.9% or D-PBS (gentamicin supplemented). 2 epididymides were transferred into a petri dish (ɸ 35mm) containing 1ml NaCl 0,9% or D-PBS which were then punctured by a sterile needle to release the sperm into the solution. Amount of sperms were determined When the parameter α and β was estimated by using hemocytometer. The final sperm concentramaximum likelihood-based method: tion ranged about 2x106 sperm/ml [5]. n Evaluate the survival rate of sperm n (αˆ + βˆ xi ) y ( e ) = The survival rate of sperm can be evaluated i   i 1 =i 1 by morphological observation or via staining. = n After staining, survival and death sperm were n ˆ  counted. xi yi = x i (e(αˆ + β xi ) )  i 1 =i 1 • Morphological observation: the morpho-= and logical good-quality sperms are the sperms can swim strong and straight, is not deformed, and  pˆ ( y | x) = eαˆ + βˆ x its head is bright resource [4, 6].  αˆ + βˆ xi • Staining observation: single staining (0.5%  ˆ p e βˆ ( xi − x0 ) i Eosin dye) or double staining (mixture of 1% Eo= = = RR x x e ( | )  0 i αˆ + βˆ x0 sin and 10% Nigrosin). The survival sperms are ˆ p e 0  not stained; the death sperm is stained red [4, 6]. The survival rate of spermatozoa was calculated pˆ ( y | x) : Predicted the survival rate follow x every replicate by counting in 100 spermatozoa. RR( xi | x0 ) : Risk ratio of the survival rate with x i Preservation of spermatozoa versus x0 The survival rate of sperms during being preserved at 4oC, 10oC and room temperature (26oC)

∑ ∑

СЕРИЯ

Физиология

∑

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van

EFFECT OF TIME AND TEMPERATURE ON THE SURVIVAL RATE OF MOUSE SPERM (MUS MUSCULUS VAR. ALBINO) IN SHORT-TERM PRESERVATION WITHOUT CRYOPROTECTANT AGENTS

Results and discussions Comparative results of evaluated methods about the survival rate The survival rate of sperms during preservation in all tests were evaluated by three methods (morphological observation, single staining

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Сетевой научно-практический журнал

and double staining). In 36 tests obtained, we randomly selected 2 tests in two solutions and two different temperature at 0 hour in order to examine the differences among three evaluated methods. The results are shown in table 1.

The survival rate of sperms were evaluated by three methods in 2 tests

Table 1

Tests I

Assessable Methods

The survival rate (%) 59.75± 2.45ac (239/400)

Morphology

II The survival rate (%) 67.50 ±2.34a (270/400)

95% CI (%) 54.94 - 64.56

95% CI (%) 62.91 - 72.09

Single staining

65.25±2.38ab (261/400)

60.58 - 69.92

68.75 ± 2.32a (275/400)

64.21 - 73.29

Double staining

66.50 ± 2.36b (266/400)

61.87 - 71.13

72.50 ± 2.23a (290/400)

68.12 - 76.88

a, b, c: significantly different (p < 0.05) follow column I: NaCl, 10oC, 0 hour; II: D-PBS, 26oC, 0 hour Effects of factors: solution, time and temperature on the survival rate of sperms after short-term preservation Based on the results shown in table 1, we choose the single staining method to present the effect of three examined factors on the survival rate of sperms after short-term preservation as in table 2. Table 2 The survival rate of sperms in examined tests

In the first test, the survival rates of sperms are significantly different between morphology and double staining methods (p = 0.049). In the second test, all three evaluated methods are not statistically significantly different (p > 0.05). This results showed that we can use all methods to evaluate the survival rate of sperms.

Time (hour)

The survival rate (%)

4oC 65.25±2.38 (261)

NaCl 10oC 65.25±2.38 (261)

26oC 65.25±2.38 (261)

4oC 68.75±2.32 (275)

D-PBS 10oC 68.75±2.32 (275)

26oC 68.75±2.32 (275)

44.75±2.49 (179)

54.75±2.49 (219)

39.50±2.44 (158)

54.50±2.49 (218)

54.00±2.49 (216)

40.05±2.45 (162)

21.25±2.05 (85)

18.00±1.92 (72)

17.50±1.90 (70)

29.25±2.27 (117)

25.75±2.19 (103)

18.50±1.94 (74)

12.50±1.65 (50)

12.00±1.62 (48)

11.25±1.58 (45)

19.50±1.98 (78)

17.50±1.90 (70)

12.50±1.65 (50)

4.75 ±1.06 (19)

7.50 ±1.32 (30)

5.00 ±1.09 (20)

9.25±1.45 (37)

8.50±1.39 (40)

5.50±1.14 (22)

1.75 ± 0.66 (7)

4.25 ± 1.01 (17)

3.75 ± 0.95 (15)

5.25±1.12 (21)

3.50±0.92 (14)

1.25±0.56 (5)

СЕРИЯ

Физиология

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van

EFFECT OF TIME AND TEMPERATURE ON THE SURVIVAL RATE OF MOUSE SPERM (MUS MUSCULUS VAR. ALBINO) IN SHORT-TERM PRESERVATION WITHOUT CRYOPROTECTANT AGENTS

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Effect of solution factor As shown in table 3, the survival rate of sperms after preservation was affected by the changes of solution factor, the difference is statistically significant (p = 0.0002). Specifically, when NaCl 0.9% was replaced by D-PBS, the survival rate of sperms increased 1.126 times (equivalent 12.6%, 95% confidence interval, change in the range from 5.7% to 20%). This can be explained that D-PBS solution has much more components than NaCl 0,9% solution. D-PBS solution consist of NaCl, KCl, KH2PO4, Na2HPO4, MgCl2.6H2O, CaCl2, while NaCl 0,9% solution is only sodium chloride in water. According to Van Thuan et al. [10], mouse sperm could be preserved for 2 months in a high-salt solution, especially, in-

creasing the concentration of NaCl in KSOMAA medium. When sperms are placed in a high concentration of NaCl, water is drawn out of the cell until equilibrium is reached [3, 10]. When epididymides were stored in salt at room temperature, about 30% of water was lost within 1 day, while epididymides were stored in glucose, about 70% of the water was lost within 1 day with no further changes [7]. So, oocyte activation capacity of spermatozoa after storage in the cauda epididymidis in salt or sugar at room temperature in 1 week, the percentage of surviving oocytes after injected was from 70% to 71%. Our results showed that the survival rate of sperms stored in D-PBS solution is higher than those in NaCl 0,9% solution. Table 3 Effect of solution on the survival rate of sperms

Predictor Constant - α Solution - β

The estimated coefficient

p-value

Risk Ratio

95% CI

-1.377 0.119

< 2e-16 0.0002

1.126

1.057 - 1.200

Effect of temperature factor The survival rate of sperms after preservation was affected by the changes of temperature factor, the difference is statistically significant (p = 2.27e5 ) (table 4). Specifically, the temperature was increased 1oC, the survival rate of sperms was decreased 0.993 times (equivalent 0.7%, 95% confidence interval, change in the range from 0.4% to 1.1%). Sankai et al. [8] reported that sperm motility decreased with increasing storage temperature (5-20oC). The decrease was significant difference between 15 and 20oC, suggesting that a

change in the metabolism activity of spermatozoa occurs between 15 and 20oC and that some factors are involved in the maintenance of sperm motility at the different temperature. Spermatozoa stored at 5oC had bent tails, possibly due to damage to the plasma membranes and to the spermatozoa’s hardening in the phospholipid by exposure to the low temperature [8]. Moreover, Sankai et al. indicated that when spermatozoa stored at 10oC are used for IVF and embryo transfer, the delivery rate might be higher than when spermatozoa are stored at 5oC. Table 4 Effect of temperature on the survival rate of sperms

Predictor Constant - α Temperature - β

The estimated coefficient

p-value

Risk Ratio

95% CI

-1.218 -0.007

< 2e-16 2.27e-5

0.993

0.989 - 0.996

Many studies also confirmed that 4°C is an optimal temperature for the preservation of freezedried spermatozoa [2, 3]. Our results showed that the survival rate of sperms stored at 4oC or 10oC is higher than those at 26oC (Table 2). Based on the results obtained, we plotted a chart СЕРИЯ

to predict the survival rate of sperms in response to change in temperature (Fig 1). Using this chart, we can predict the survival rate of sperms during short-term preservation in NaCl 0.9% or D-PBS solution corresponding to the change of temperature in the range from 4oC to 26oC.

Физиология

EFFECT OF TIME AND TEMPERATURE ON THE SURVIVAL RATE OF MOUSE SPERM (MUS MUSCULUS VAR. ALBINO) IN SHORT-TERM PRESERVATION WITHOUT CRYOPROTECTANT AGENTS

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Predicted the survival rate = exp(-1.218 - 0.007 * 'Temperature') Predicted the survival rate

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van

0.7

Regression 95% CI

0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

Temperature Fig 1. A chart predict the survival rate of sperms in response to change in temperature Effect of time factor The results in table 5 showed that the survival rate of sperms after preservation was affected by the changes of time factor, the difference is statistically significant (p < 2e-16). Specifically, every one hour extended, the survival rate of sperms was decreased 0.932 times (equivalent 6.8%, 95% confidence interval, change in the range from

6.6% to 7.1%). This can be explained that two preservation solutions do not include any nutrition ingredients (only different kinds of salt), so the longer the storage time is, the more the survival rate of sperms is. Sato et al. [9] reported that the percentage of spermatozoa motility decreased when the preservation extended in the longer time (TYH media at 22oC and room temperature).

Effect of time on the survival rate of sperms The estimated coefficient

p-value

Constant - α

-0.327

< 2e-16

Time - β

-0.071

< 2e-16

Predictor

This result is consistent with the theory and previously reported studies. Based on the results obtained, we plotted a chart to predict the survival rate of sperms in response to the change of time (Fig 2). Using the chart, we can calculate the survival rate of sperms during short-term storage in NaCl 0.9% or D-PBS solution corresponding to the time prolongated. СЕРИЯ

Table 5

Risk Ratio

95% CI

0.932

0.929 - 0.934

For example, the figure 2 shows that the survival rate of sperms was predicted at 0 hour is 72% and at 9.76 hours is 36%. This means the lethal concentration and time (LCt50) to mouse sperm in short-term storage is 10 hours in D-PBS or NaCl 0,9% solution without cryoprotectant agents.

Физиология

EFFECT OF TIME AND TEMPERATURE ON THE SURVIVAL RATE OF MOUSE SPERM (MUS MUSCULUS VAR. ALBINO) IN SHORT-TERM PRESERVATION WITHOUT CRYOPROTECTANT AGENTS

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Predicted the survival rate of sperm = exp(-0.327 - 0.071 * 'Time') Predicted the survival of rate

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van

9.76

0.8

Regression 95% CI

0.7 0.6 0.5 0.4

0.36

0.3 0.2 0.1 0.0 0

Time (hour)

Fig 2. A chart predict the survival rate of sperms in response to change in time Effect of three evaluated factors The survival rate of sperms after preservation was affected by the changes of three factors (solution, temperature, time), the difference is very statistically significant (p < 0.001) (table 6).

When all three factors examined at the same time, the results are highly similar to those obtained when examined every factor separately. Consequently, an equation was set to predict the survival rate of sperms follow effect of examined factors:

A: Predicted the survival rate Apply the equation to the data collected from our analysis, in the case of: α = -0.291; β1 = 0.119; β2 = -0.007; β3 = -0.071; Solution = 0 for NaCl; Solution = 1 for D-PBS, we have a prediction equation for our experiments

Effect of three evaluated factors on the survival rate of sperms

Table 6

Constant - α

The estimated coefficient -0.291

Solution - β1

0.119

0.0002

1.126

1.057-1.200

Temperature - β2

-0.007

2.27e-05

0.993

0.989-0.996

Time - β3

-0.071

< 2e-16

0.932

0.929-0.934

Predictor

СЕРИЯ

p-value

Risk Ratio

95% CI

< 2e-16

Физиология

Huyen Nguyen Thi Thuong, Ly Dao Thi My, Phung Nguyen Quang, Vi Le Thi Tuong, Quan Ke Thai, Tri Truong Van

EFFECT OF TIME AND TEMPERATURE ON THE SURVIVAL RATE OF MOUSE SPERM (MUS MUSCULUS VAR. ALBINO) IN SHORT-TERM PRESERVATION WITHOUT CRYOPROTECTANT AGENTS

Conclusion The short-term preservation of mouse sperms can be implemented at 4oC or 10oC in NaCl 0,9% or D-PBS solution without cryoprotectant agents. The survival rate of sperm in D-PBS solution is higher than those in NaCl 0,9% solution.

References: 1. Kaneko, T. and N. Nakagata (2005), “Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa”, Comp Med, 55(2): p. 140-4. 2. Kaneko, T. and N. Nakagata (2006), “Improvement in the long-term stability of freezedried mouse spermatozoa by adding of a chelating agent”, Cryobiology, 53(2): p. 279-82. 3. McGinnis, L.K., L. Zhu, J.A. Lawitts, S. Bhowmick, M. Toner, and J.D. Biggers (2005), “Mouse sperm desiccated and stored in trehalose medium without freezing”, Biol Reprod, 73(4): p. 627-33. 4. Minh Le Thi and Huyen Nguyen Thi Thuong (2008), Thực hành Sinh lí người và động vật, NXB Đại học Sư phạm (lưu hành nội bộ). 5. Nagy, A., M. Gertsenstein, K. Vintersten, and R. Behringer (2003), Manipulating the mouse embryo: A laboratory manual, Third ed, Cold Spring Harbor Laboratory Press, New York, 764.

Сетевой научно-практический журнал

The lethal concentration and time (LCt50) to mouse sperm in short-term preservation is 10 hours in D-PBS or NaCl 0,9% solution without cryoprotectant agents.

6. Ngoc Phan Kim and Phuc Pham Van (2010), Công nghệ Sinh học trên người và Động vật, NXB Giáo dục Việt Nam. 7. Ono, T., E. Mizutani, C. Li, and T. Wakayama (2010), “Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature”, Zygote, 18(3): p. 245-56. 8. Sankai, T., H. Tsuchiya, and N. Ogonuki (2001), “Short-term nonfrozen storage of mouse epididymal spermatozoa”, Theriogenology, 55(8): p. 1759-68. 9. Sato, M. and A. Ishikawa (2004), “Room temperature storage of mouse epididymal spermatozoa: exploration of factors affecting sperm survival”, Theriogenology, 61(7-8): p. 1455-69. 10. Thuan Van , N., S. Wakayama, S. Kishigami, and T. Wakayama (2005), “New preservation method for mouse spermatozoa without freezing”, Biol Reprod, 72(2): p. 444-50. 11. Tsuchiya, H., N. Ogonuki, T. Kuwana, T. Sankai, and K. Kanayama (2001), “Short-term preservation of mouse oocytes at 5 degrees C”, Exp Anim, 50(5): p. 441-3.

DATA ABOUT THE AUTHOR: Huyen Nguyen Thi Thuong1*, Ly Dao Thi My1, Phung Nguyen Quang1, Vi Le Thi Tuong1, Quan Ke Thai2, Tri Truong Van1 1 University of Education, Ho Chi Minh city, Vietnam 2 Saigon University, Ho Chi Minh city, Vietnam *Corresponding author: Huyen Nguyen Thi Thuong. Email: huyenntth@hcmup.edu.vn

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Физиология

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Физиология

А У Ч Н ЫЙ РЕЗУЛЬТАТ

УДК 576-08

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

Abstract

ead (Pb) is a toxic metal and and can cause variety of disorders and effect on neu-ronal function and neurodevelopment. Using zebrafish as a model, the aim of this study was to evaluate the effects of concentrations of Pb2+ on the life of zebrafish larvae (from 1 to 7 days old). About 3-5 minutes after mating, collecting embryos and embryos were continuously exposed to Pb2+ at the different concentrations: 0 μg/L, 20 μg/L, 40 μg/L, 60 μg/L, 80 μg/L, 100 μg/L, 120 μg/L, 140 μg/L in embryo Hank medium. After hatching, larvae were transferred to larval Hank medium supplemented corresponding concentrations of Pb2+. The results show: (i) in the different examined concentrations of Pb2+, the minimum concentration of Pb2+ affected the survival rate of larval zebrafish is 40µg/l; (ii) at every examined concentrations of Pb2+, the survival rate of larval zebrafish was affected significantly on the 6th and 7th days. Based on the results obtained, we set up a equation to predict the survival rate of zebrafish larvae using two factors: concentrations of Pb2+ and time of culture. The lethal concentration and time to larval zebrafish stage is 7 cultured days in 68.9 µg/l concentraion of Pb2+.

ey words: lead, zebrafish or Dianio rerio, larvea, heavy metal.

The development of the industry and agriculture leads to the increasing of environ-mental pollution. One of the cause of the pollution is heavy metals pollution which is considered an urgent problem, especially for the aquatic ecosystem. When heavy metals such as mercury (Hg), cadmium (Cd), arsenic (As), and lead (Pb)... accumulate in water, they can lead to the dangers in aquatic animals, hence the humans’ health [4]. Lead is a toxic metal and and can cause a variety of disorders and effect neuronal function and neurodevelopment (Neal et al., 2011 [10]; Rice et al., 2011 [12]). In experimental animals, acute lead exposure can result in neurotoxic effects such as, behavioral abnormalities, learning impairment, hearing loss, and impaired cognitive functions [3]. The use of fish as bioindicators in order to evaluate the heavy metal pollution in aquatic environments have been reported (Ebrahimi et al.,

2010 [4]). The study of the effects of Pb2+ on the living organisms using zebrafish (Danio rerio) as model organism have been implemented by scientists all over the world. In Vietnam, the influences of Pb2+ was evaluated primarily by the chemical or physical methods, there is not an accurate evaluation on the growth of aquatic animals, especially on vertebrate animals. Fishes at embryo and larval stage have the highest sensitivity in their life [1, 6]. The accumulation of heavy metal in the body or organs of embryos affect to the growth of fishes. This study used infected zebrafish embryos to examine the affect of lead on larvae. Material and method Zebrafish maintenance and mating The Zebrafish (about 2 months old) were maintained in light controlled room (14-hours light and 10-hours dark cycle) at room tem-

№2 2014

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

perature [5, 13, 16] until getting sexual maturity. Males and females were kept separately. All experimental procedures were carried out in the Laboratory of Anatomy - Human and Animal physiology, the University of Education in Ho Chi Minh city. The glass mating tanks (60 mm diameter) were filled two-thirds full, with marbles at the bottom to make racks for embryo clinging and to prevent embryo cannibalism. Males and females at a ratio of 1:2 are separated by a transparent bulkhead in a tank with a light-dark cycle condition right before mating. [14, 15] Collect embryo and treat with different concentrations of Pb2+ Embryo and larval Hank media (pH 7-7.5) were used in embryo and larval zebrafish experiments, respectively. Pb(NO3)2 was dissolved in Hank medium at stock concentrations of 10 mg/l, and then diluted to final concentrations in embryo Hank media at the stages indicated [14]. About 3-5 minutes after mating, fishes were transferred to a new tank. Bottom of the mating tank would be checked quickly for the present of embryos. After the removal of marbles, embryos from mating tanks would transferred to glass beaker by siphon. Only morphological good-quality embryos (morphological life, homogeneous cytoplasm, not mis-shapen) were used for experiments. All embryos were incubated at room temperature, pH 7-7.5. Embryos were continuously exposed to Pb2+ at the following examined concentrations: 0 μg/L, 20 μg/L, 40 μg/L, 60 μg/L, 80 μg/L, 100 μg/L, 120 μg/L, 140 μg/L in the embryo Hank medium. Four replicates (n = 4), each of which containing 20 embryos in a 60-mm diameter glass Petri dishes, were cultured in glass beaker with a volume of 200ml embryo Hank medium with supplementation of Pb2+ at examined concentrations (totally 80 embryos for every concentration). After hatching, larvae were transferred to larval Hank medium with supplementation of Pb2+ at corresponding concentrations [2, 3, 5, 12]. Evaluate the survival of larvae Concentrations of Pb2+ and pH of the larval Hank medium were kept constantly. The survial/death rate of larvae was calculated every 24 hours, to evaluate the survival rate of larval СЕРИЯ

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zebrafish depending on the correlation between time and concentration of Pb2+. Based on the collected data, an equation was set up to predict 50% lethal threshold of zebrafish larvae using these factors. Statistical Analysis All data obtained from this study were calculated by Minitab 16, R software. Data are given as the mean ± SE. For all statistical tests, differences were considered statistically different at p < 0.05. Use of logistic regression analysis method with Poission regression model to analyze the correlation between the survival rate of larval zebrafish and examined factors. This model is µ  log  i = α + β xi  Ni  ; this means log of a function: the survival rate of larval zebrafish is a function depend on x factor. When the parameter α and β was estimated by maximum likelihood-based method: n n (αˆ + βˆ xi ) y ( e ) = ∑ ∑ i   i 1 =i 1 = n n  x y = x (e(αˆ + βˆ xi ) ) ∑ i i i ∑ i 1 =i 1 =

and

 pˆ ( y | x) = eαˆ + βˆ x  ˆ  pˆ i eαˆ + β xi βˆ ( xi − x0 ) = = = RR x x e ( | )  0 i αˆ + βˆ x0 ˆ p e 0  pˆ ( y | x) : Predicted the survival rate follow x RR( xi | x0 ) : Risk ratio of the survival rate with

xi versus x0

Results and discussion The survival of larval zebrafish at the examined time and concentrations of Pb2+ The zebrafish larvae were cultured in embryo Hank medium with the 7 examined concentrations of Pb2+ and control group. The survival rate of larvae by cultured every day shown in table 1.

Физиология

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

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Table 1

The survival rate of larval zebrafish by days (%) Pb concentration (µg/l)

62.50± 5.41 (50/80)

61.25± 5.45 (49/80)

55.00± 5.56 (44/80)

53.75± 5.57 (43/80)

51.25± 5.59 (41/80)

70.00± 5.12 (56/80)

66.25± 5.29 (53/80)

62.50± 5.41 (50/80)

60.00± 5.48 (48/80)

63.75± 5.37 (51/80)

57.50± 5.53 (46/80)

33.75± 5.29 (27/80)

27.50± 4.99 (22/80)

26.25± 4.92 (21/80)

22.50± 4.67 (18/80)

21.25± 4.57 (17/80)

77.50± 4.67 (62/80)

58.75± 5.50 (47/80)

47.50± 5.58 (38/80)

38.75± 5.45 (31/80)

37.50± 5.41 (30/80)

26.5± 4.92 (21/80)

25.00± 4.84 (20/80)

61.25± 5.45 (49/80)

52.50± 5.58 (42/80)

51.25± 5.59 (41/80)

45.00± 5.56 (36/80)

37.50± 5.41 (30/80)

100

62.50± 5.41 (50/80)

58.75± 5.50 (47/80)

57.50± 5.53 (46/80)

55.00± 5.56 (44/80)

30.00± 5.12 (24/80)

120

75.00± 4.84 (60/80)

71.25± 5.06 (57/80)

68.75± 5.18 (55/80)

57.50± 5.53 (46/80)

51.25± 5.59 (41/80)

43.75± 5.55 (35/80)

140

76.25± 4.76 (61/80)

73.75± 4.92 (59/80)

58.75± 5.50 (47/80)

53.75± 5.57 (43/80)

35.00± 5.33 (28/80)

31.25± 5.18 (25/80)

30.00± 5.12 (24/80)

Examining the fluctuations of the survival rate of larvae by days in medium without Pb2+ Based on the results of table 1, we examined the fluctuations about the survival rate of larvae by days in medium without Pb (control group). The results shown in table 2. Table 2 2+ Effect of time on the survival rate of larvae in medium without Pb The estimated coefficient

p-value

Constant - α

-0.424

0.0005

Day - β

-0.035

0.2080

Predictor

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Физиология

Risk Ratio

95% CI

0.965

0.914 - 1.020

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

As shown in table 2, the survival rate of larvae in control group had varied during cultured days, but the difference is not statistically significant (p = 0.2080). Specifically, the survival rate of larvae was decreased 0.965 times (equivalent 3.5%) every day (from 62.50% on the first day to 51.25% on the 7th day, see table 1). It could be infered that this variation occurred randomly (p = 0.2080); after hatching, the fish had been developing from embryo to larvae, and not yet adapted to new conditions; in the mean time, larvae subsisted largely on yolk-sac reserves until the onset of exogenous feeding. After 5-6 days post fertilization (dpf), duration for the completely development of the functional

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digestive system, the yolk-sac would gradually exhausted (Kimmel et al., 1995 [8]), and disappeared on the 7th day (Jardine and Litvak, 2003 [7]). From this point on (and preferably before), the larvae must be self-feeding. In the life time of fish, larval stage (especially, the first larval stage) is often the more sensitive than adult stage (Arufe et al, 2004 [1], Hwang et al. 1995 [6]). Thus, our experimental results showed that the survival rate of larvae in control group after cultured days decreased slightly. Based on the results obtained, we plotted a chart to predict the survival rate of larvae in cultured medium without Pb2+ (Fig 1).

Predicted the survival rate = exp(-0.424 - 0.035*'Day')

Predicted the survival rate

0.66

Regression 95% C I

0.64 0.62 0.60 0.58 0.56 0.54 0.52 0.50 1

Day Fig 1. A chart for prediction of the survival rate of larvae in medium without Pb2+ Examining of fluctuation of the survival of larvae by cultured days in medium with Pb2+ Based on the results of table 1, we evaluated of fluctuation of the survival of larval zebrafish by cultured days in medium with the examined concentrations of Pb2+. The results shown in table 3. Table 3 Effect of time on the survival rate of larvae in medium with the examined concentrations of Pb2+ Pb concentration (µg/l) 20 40

Predictor

The estimated coefficient

p-value

-0.306 -0.026 -0.265 -0.210

0.0071 0.3121 0.0541 1.46e-08

Constant - α Day - β Constant - α Day - β СЕРИЯ

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Risk Ratio

95% CI

0.974

0.926-1.025

0.810

0.753-0.871

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

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As shown in table 2, at lead concentration of 20 µg/l, the survival rate of larval zebrafish decreased after three days cultured. Specifically, the survival rate of larvae was decreased 0.974 times (equivalent 2.6%) every day cultured (from 70.00% on the first, second and third days to 60.00% on the 7th day, see table 1). And, this variation is due to random (p = 0.3121). In other words, the 20 µg/l concentration of Pb2+ is not lethal threshold of larvae zebrafish. Meanwhile, at lead concentration of 40 µg/l, the survival rate of larval zebrafish has statistically significant difference by cultured days (p = 1.46 e-08). Specifically, the survival rate of larvae was decreased 0.810 times (equivalent 19%, 95% confidence interval, change in the range from

12.9% to 24.7%) by cultured everyday (from 63.75% on the first day to 21.25% on the 7th day, see table 1). The survival rate of larvae in concentrations of Pb2+ (60, 80, 100, 120 and 140 µg/l) also give similar results. This meant that the minimum concentration of Pb2+ in the tests effecting on the survival rate of larval zebrafish is 40 µg/l. Our analysis showed the same results reported by Duo et al. (2011) [3] and Rice et al. (2011) [12]. According that, when embryo stages were exposed to low concentrations of Pb2+, they were not affected the activites and behaviors of fish later. Based on the results obtained, we can plot a chart predict the survival rate of larvae by cultured days in Hank medium with 20 µg/l Pb2+ (Fig 2) and 40 µg/l Pb2+ (Fig 3).

Predicted the survival rate

Predicted the survival rate = exp(-0.306 - 0.026*'Day') 0.74

Regression 95% CI

0.72 0.70 0.68 0.66 0.64 0.62 0.60 1

Day Fig 2. A chart predict the survival rate of larvae in medium with 20 µg/l Pb2+

Predicted the survival rate = exp(-0.265 - 0.210*'Day')

Predicted the survival rate

0.7

Regression 95% CI

0.6 0.5 0.4 0.3 0.2 0.1 1

Day Fig 3. A chart predict the survival rate of larvae in medium with 40 µg/l Pb2+ СЕРИЯ

Физиология

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

Examining of the effect of lead concentrations on the survival rate of larvae by every cultured day Base on the result of table 1, we fixed the timelines by every day (from the first day to the 7th day, 7 timelines, respectively), then evaluated to the fluctuation of the survival rate of larval zebrafish following the increasing the concentration of Pb. The results shown in table 4. The results in table 4 shown that the increasing the concentration of Pb2+ didn’t affect the

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fluctuation of the survival rate of larval zebrafish on the first day (p = 0.399). As mentioned above (section 3.2), on the first day after hatching, larvae subsist largely on yolk-sac, so the embryo viability remained. Thus, when the larvae were exposed to Pb2+ (20, 40, 60, 80, 100, 140 µg/l) in larval Hank medium, Pb2+ could not penetrate into the fish’s body in order to adversely impact in them [9]. Table 4

Effect of lead concentrations on the survival rate of larvae at the examined time Day 1 5 6 7

Predictor Constant - α Day - β Constant - α Day - β Constant - α Day - β Constant - α Day - β

The estimated coefficient -0.439 0.001 -0.691 -0.002 -0.749 -0.003 -0.781 -0.003

Analyzing the results in table 1 on the days (2 , 3rd, 4th and 5th), we also get similar results on the first day. This show the increasing the concentration of Pb2+ didn’t affect the fluctuation of the survival rate of larval zebrafish on the cultured days (from the 1st to the 5th day). Thus, on the early days (from the 1st to the 5th day), the fluctuation of the survival rate of larval zebrafish is not affected clearly by the increasing the concentration of Pb2+. On the 6th and the 7th day, the survival rate of larval zebrafish decreased when the concentration of Pb2+ increased, the difference is statistically significant (p < 0.05). Specifically, the survival rate of larvae was decreased 0.997 times when the concentration of Pb2+ increased 1µg/l (equivalent 0.3%, 95% confidence interval, change in the range from 0.1% to 0.6%) on the 7th day (from 60.00% in 20 µg/l concentration to 30.00% in 140 µg/l concentration, see table 1). This can be explained in the way that: (i) on the 6th and the 7th days, Pb2+ penetrated into the fish’s body via gills, skin and mouth to accumulate and nd

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p-value 7.5e-07 0.399 4.97e-11 0.085 6.1e-12 0.048 2.12e-12 0.031

Risk Ratio

95% CI

1.001

0.999 - 1.003

0.998

0.995 - 1.000

0.997

0.995 - 0.999

0.997

0.994 - 0.999

affect to the survival rate of larval zebrafish. According to Peterson et al., 2010 [11], there are 30 genes involving to the development of the fish’s body which were mutated during the Pb2+ exposure time; (ii) Moreover, the zebrafish cultured conditions have a lots of disadvantageous factors such as light, noise, the concentration of O2 or CO2 which can stress zebrafish. Our results of experiments consistent with publish of Hwang et al. 1995 [6], in which they have demonstrated the larvae are the most sensitive stage for the fish life cycle, the sensitiveness of early larval stage increase when the environment is pollution by heavy metal (such as lead). The lavae usually do not have gills, their skin have permeability to respise and transfer ions. Thus, the toxic substances can penetrate into fish’s body through their skin which causing to change disadvantage for fish, especially, larval stage [9, 13]. During the experiment, we saw a few malformations (edema in the head or heart) of the larval zebrafish (Fig 5).

Физиология

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

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Fig 5. A few malformations of the larval zebrafish (X40) The larvae was edema in the head (A, B, D) or heart (C) - The position of larvae was edema - measure: 500µm

Predicted the survival rate = exp(-0.439 + 0.001 *'Concentration')

Predicted the survival rate

0.80

Regression 95% C I

0.75

0.70

0.65

0.60 0

100

120

140

Concentration (µg/l )

Fig 4. A chart for prediction of the survival rate of larvae following concentration of Pb2+ (on the 1st day) СЕРИЯ

Физиология

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

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Predicted the survival rate = exp(-0.781 - 0.003*'Concentration')

Predicted the survival rate

Regression

0.6

95% C I

0.5

0.4

0.3

0.2 0

100

120

140

Concentration (µg/l )

Fig 5. A chart for prediction of the survival rate of larvae following concentration of Pb2+ (on the 7th day) Based on the results obtained, it can be concluded that the time and the concentration of Pb2+ factors interacted each other to affect the fluctuation of the survival/death rate of zebrafish larvae. Examining the effect of the interaction between time and concentration of Pb2+ on the survival rate of larval zebrafish Based on the resluts of table 1, we evaluated the fluctuation of the survival rate of zebrafish

larvae by affecting the interaction of time and concentration of Pb2+ factors according to the Poission regression model which is stated as following: Log(µi/Ni) ~ (Time * Concentration) Or: Log(µi/Ni) = α + β1*Time + β2*Concentration + β3*Time* Concentration The results shown in table 5. Table 5

Effect of interaction between time and concentration of Pb2+ on the survival rate of zebrafish larvae The estimated coefficient

p-value

Risk Ratio

95% CI

Constant - α

-0.419

1.5e-07

Day - β1

-0.058

0.0025

0.944

0.909 - 0.980

Concentration - β2

0.002

0.0136

1.002

1.000 - 1.004

interaction - β3

-0.001

0.0014

0.999

0.998 - 0.999

Predictor

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Физиология

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

The results in table 5 shown the fluctuation of the survival rate of zebrafish larvae are affected during the time, concentration and interaction between the time and the concentration factor, all the differences are statistically significant (p < 0.05). Consequently, we set up a equation to predict the survival rate of larval zebrafish follow effect of factors: Predicted the survival rate = exp(α + β1*Time + β2*Concentration + β3*Time*Concentration)

Apply the equation to the data collected from our analysis, in the case of α = -0.419; β1 = -0.058; β2 = 0.002; β3 = -0.001, we have a prediction equation: Predicted the survival rate = exp(-0.419 - 0.058*Time + 0.002*Concentration – 0.001*Time*Concentration) (1) Using the prediction equation (1), we can plot a chart to predict the survival rate of larvae following interaction between Pb2+ concentration and culture time (Fig 6); and can calculate LCt50

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(lethal concentration and time) of Pb2+ to the larval zebrafish stage (7 days) as follows: The survival rate estimates on the first day in Hank medium without Pb2+ is: Pr0 (time = 1, Pb2+ = 0) = exp(-0.419 – 0.058*1) = 0.621 The survival rate estimates on the 7th day in Hank medium without Pb2+ is: Prx (time = 7, Pb2+ = X) = exp(-0.419 – 0.058*7 + 0.002*X – 0.001*7*X) = exp(-0.825 - 0.005*X) LCt50 means: Prx/ Pr0 = 0.5 → exp(-0.825 0.005*X) / 0.621 = 0.5 → -0.005*X = Ln(0.3105) + 0.825 → X = 68.9 µg/l Pb2+ So, at 68.9 µg/l concentraion of Pb2+, the survival rate of larval zebrafish will decrease 50% by cultured 7 days. This means the lethal concentration and time to larval zebrafish stage is 7 cultured days in 68.9 µg/l concentraion of Pb2+.

Fig 6. A chart predict the survival rate of larvae following interaction between concentrations of Pb2+ and culture time Conclusion Among the examined concentrations of Pb2+, the minimum concentration of Pb2+ affected to the survival rate of larval zebrafish is 40µg/l. The survival rate of larval zebrafish was affected significant on the 6th and 7th cultured days at a certain concentration of Pb2+. СЕРИЯ

An equation for prediction of the survival rate of zebrafish larvae was set using two factors: concentrations of Pb2+ and culture time. The lethal concentration and time to larval zebrafish stage is 7 cultured days in 68.9 µg/l concentraion of Pb2+.

Физиология

Huyen Nguyen Thi Thuong, Quan Ke Thai, Vi Le Thi Tuong, Tri Truong Van, Dung Tran Thi Phuong

EXAMINING THE EFFECTS OF LEAD ON THE LIFE OF LARVAL ZEBRAFISH (1-7 DAYS OLD)

References: 1. Arufe, M.I., J. Arellano, M.J. Moreno, and C. Sarasquete (2004), “Toxicity of a commercial herbicide containing terbutryn and triasulfuron to seabream (Sparus aurata L.) larvae: a comparison with the Microtox test”, Ecotoxicol Environ Saf, 59(2): p. 209-16. 2. Braunbeck, T.a.L., E. (2006), Fish embryo toxicity assays, Umwelt Bundes Amt. Germany, p 9-12 3. Dou C., Z.J. (2011), “Effects of lead on neurogenesis during zebrafish embryonic brain development”, J Hazard Mater, 194: p. 277-82. 4. Ebrahimi, M. and M. Taherianfard (2010), “Concentration of four heavy metals (cadmium, lead, mercury, and arsenic) in organs of two cyprinid fish (Cyprinus carpio and Capoeta sp.) from the Kor River (Iran)”, Environ Monit Assess, 168(1-4): p. 575-85. 5. Graham J. Lieschke, Andrew C. Oates†, and Koichi Kawakami (2009), Zebrafish: Methods and Protocols, Methods in molecular Biology. 6. Hwang P.P., Lin S.W., and L. H.C. (1995), “Different sensitivities to cadmium in tilapia larvae (Oreochromis mossambicus, Teleostei). Arch. Environ”, Contam. Toxicol 29: p. 1-7. 7. Jardine, D., Litvak, M.K. (2003), “Direct yolk sac volume manipulation of zebrafish embryos and the relationship between offspring size and yolk sac volume”, Fish Biol., 63: p. 388–397. 8. Kimmel, C.B., W.W. Ballard, S.R. Kimmel, B. Ullmann, and T.F. Schilling (1995), “Stages of embryonic development of the zebrafish”, Dev Dyn, 203(3): p. 253-310.

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9. Lawrence, C. (2007), “The husbandry of zebrafish (Danio rerio): A review”, Aquaculture, 269: p. 1-20. 10. Neal, A.P., P.F. Worley, and T.R. Guilarte (2011), “Lead exposure during synaptogenesis alters NMDA receptor targeting via NMDA receptor inhibition”, Neurotoxicology, 32(2): p. 281-9. 11. Peterson, S.M., J. Zhang, G. Weber, and J.L. Freeman (2010), “Global gene expression analysis reveals dynamic and developmental stage-dependent enrichment of lead-induced neurological gene alterations”, Environ Health Perspect, 119(5): p. 615-21. 12. Rice C., G.J.K., Zalewski K., Weber D. N. (2011), “Developmental lead exposure causes startle response deficits in zebrafish”, Aquat Toxicol, 105(3-4): p. 600-8. 13. Steve F. Perry, Marc Ekker, Anthony P. Farrell, and Colin J. Brauner (2010), Zebrafish, First ed, Elsevier, United States of America, 452. 14. Westerfield, M. (2000), The zebrafish book: A guide for the laboratory use of zebrafish (Brachydanio rerio), ed. E. 3rd, University of Oregon Press, Institute of Neuroscience, USA. 15. Westerfield M (1995), The zebrafish book. 5th edition; A guide for the laboratory use of zebrafish (Danio rerio), Eugene, University of Oregon Press. Paperback. 16. Westerfield M. (2007), The zebrafish book, 5th ed, A guide for the laboratory use of zebrafish (Danio rerio), Eugene, University of Oregon Press. Paperback.

DATA ABOUT THE AUTHOR: Huyen Nguyen Thi Thuong1*, Quan Ke Thai2, Vi Le Thi Tuong1, Tri Truong Van1, Dung Tran Thi Phuong1 1 University of Education, Ho Chi Minh city, Vietnam 2 Saigon University, Ho Chi Minh city, Vietnam *Corresponding author: Huyen Nguyen Thi Thuong. Email: huyenntth@hcmup.edu.vn

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УДК 576.322.2

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

Abstract

ased on checklist, the keys to the identification updating with the new name of species according to Sang Nguyen Van et al author of the book “Herpetofauna of Vietnam” publisher Chimaira published in 2009 and the biological and ecological data of 120 species of amphibian and reptiles known from the herpetofauna in three western provinces of South - Eastern region, South Vietnam was announced by Hoa Pham Van in 2005 to build the web-based tool for identification of species from this herpetofauna. It is the first online tool used with vietnamese interface for identifying 118 species from this herpetofauna. Therefore, it can help for users with a means to quickly and easily identify and lookup them than when they used the keys to the identification of amphibian and reptiles species printing in the printout version. This tool can be accessed via internet at http://zoology.vietbiodata.net.

ey words: Amphibia, DAR, database for Amphibian and Reptiles, Reptiles, South-Eastern region.

Three western provinces (BINH DUONG, BINH PHUOC, TAY NINH) of South-Eastern region in the South Vietnam is located in 10025’12017’N and 105048’-107028’E. Natural area of it is 13566 square kilometers. Year average temperature from 26.20C to 26.90C. Average annual rainfall from 1813.1 to 2469.2mm and humidity is from 78 to 79 percent. Their vegetation is very diverse with evergreen forest, semi-evergreen forest, mixed forest of timber bamboo, grassland and scrub. Studies on amphibian and reptiles species in the South Vietnam were conducted from 19th century (1875) by number of foreign scientists. Although these studies had not been completed and systematic, they covered several places in South Vietnam’s internal and are mainly to detect species composition and distribution of amphibian and reptiles. The first research studying fully and systematically on herpetofauna of this region of the Hoa Pham Van was reported in 2005. This work has detected and recorded 88 reptile species belonging to 3 orders, 17 families

and 32 amphibian species belonging to 2 orders, 7 families. From 2005 to 2009, there have been 4 new species of lizard be discovered, described and published by some other authors in this region. As a result, the total number of species curently known from this herpetofauna is 124 species comprising 92 reptiles belonging to 3 orders, 18 families and 32 amphibian species belonging to 2 orders, 7 families. Based on the keys to identification of amphibian and reptiles and data on biology and ecology of species from herpetofauna in three western provinces of South - Eastern region, South Vietnam published by Hoa Pham Van in 2005[1], we have built the database for Amphibian and Reptiles (DAR) in the studied region which is accessible at http://zoology.vietbiodata.net. Materials and methods Data using to build database were obtained from information on the biological, ecological and taxonomic characteristics and keys to the

№2 2014

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

identification of amphibian and reptiles species from the herpetofauna in three western provinces of South - Eastern region, South Vietnam reported by Hoa Pham Van in 2005. The checklist, data on biology and ecology of species and the keys to the identification of amphibian and reptiles species from the herpetofauna in three western provinces of South - Eastern region, South Vietnam updating with the new name of species according to Sang Nguyen Van et al, [2] used to build the database of website and the web-based tool for identification of amphibian and reptiles known from this herpetofauna. Database is in-house developed and managed by MySQL Keys for identification of amphibian and reptiles in the studied region are dichotomous keys. Each step of identification has only two alternatives which are identification features on body forming, position of eyes, the presence of

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

teeth, etc… The tool was in-house developed using Perl programming language and Hypertext Markup Language (HTML). DAR is accessible at http://zoology.vietbiodata.net which is a LinuxApache-MySQL-Perl system. Result and discussion In three western provinces of South – Eastern region, South Vietnam, 124 species consist of 92 reptiles and 32 amphibian species were identified. The biological diversity in this area is very high with the representatives of many species presented in Vietnam. The diversity of reptiles is high with 92 species belonging to 3 orders, 18 families while 32 amphibian species belong to 2 orders, 7 families. Detailed information about scientific name, English name, Vietnamese name, taxonomy of each species were shown in Table 1. Biological and ecological information of each species were also recorded and stored in database for mining

List of reptiles and amphibian species presented in examined region Order number

Scientific name

English name

Vietnamese name

AMPHIBIA Linnaeus, 1758

Class AMPHIBIA

Lớp Ếch nhái

Anura FISCHER VON WALDHEIM, 1813

Order Anura

Bộ Không đuôi

I. Bufonidae GRAY, 1825

Family Bufonidae

Dutaphrynus melanostictus (SCHNEIDER, 1799)

Cóc nhà.

Ingerophrynus galeatus (GṺNTHER, 1864)

Black-spined toad, Asian common toad, Common Sunda toad. Cambodian toad.

Cóc rừng.

II. Hylidae RAFINESQUE, 1815

Family Hylidae

Họ Nhái bén

Hyla simplex BOETTGER, 1901

Annam treefrog.

Nhái bén nhỏ.

III. Megophryidae BONAPARTE, 1850

Family Megophryidae

Họ Cóc bùn

Leptobrachium pullum (SMITH, 1921)

Chapa spadefoot toad.

Cóc mày Hatxen.

Xenophrys major (Boulenger, 1908)

Anderson’s spadefoot toad.

Cóc mắt bên.

IV. Microhylidae GṺNTHER, 1858

Family Microhylidae

Họ Nhái bầu

Caluella guttulata (BLYTH, 1855)

Burmese squat frog, Blotched burrowing frog.

Ễnh ương đốm.

СЕРИЯ

Физиология

Table 1

Họ Cóc

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Glyphoglossus molossus GṺNTHER, 1869

Balloon frog, Broadlipped frog.

Nhái lưỡi.

Kalophrynus interlineatus (BLYTH, 1855)

Sumatra grainy frog, Spotted narrowmouthed frog, Northern sticky frog, Snoring frog.

Cóc đốm.

Kaloula pulchra GRAY, 1831

Banded bull frog, Malaysian narrowmouthed toad, Asiatic painted frog.

Ễnh ương thường.

Microhyla berdmorei (BLYTH, 1856)

Bermore’s narrowmouthed frog,

Nhái bầu bec mơ.

Microhyla butleri BOULENGER, 1900

Butler’s pigmy frog, Butler’s rice frog.

Nhái bầu but lơ.

Microhyla fissipes (BOULENGER, 1884)

Ornate pigmy frog.

Nhái bầu hoa, nhái dế.

Microhyla pulchra (HALLOWELL, 1861)

Guangdong rice frog.

Nhái bầu vân, ễnh ương nhỏ.

Pigmy frog.

Nhái bầu chân vịt

Deli paddy frog, Jewel pigmy frog.

Nhái bầu trơn.

V. Dicroglossidae ANDERSON, 1871

Family Dicroglossidae

Họ Ếch nhái thực

Fejervarya cancrivora (GRAVENHORST, 1829)

Marsh frog, Java wart frog, Mangrove frog, Crab-eating frog.

Ếch cua.

Fejervarya limnocharis (GRAVENHORST, 1829)

Grass frog, paddy frog, rice frog, asian pool frog.

Nhái, ngóe.

Hoplobatrachus rugulosus (WIEGMANN, 1834)

Common lowland frog, Chinese bullfrog, Taiwanese frog.

Ếch đồng, ếch, ếch ruộng.

Limnonectes kuhlii (TSCHUDI, 1838)

Kuhl’s creek frog, Bigheaded frog.

Quasipaa verrucospinosa (BOURRET, 1937)

Granular spiny frog.

Occidozyga lima (GRAVENHORST, 1829)

Green puddle frog, Rough-skinned floating frog, java frog.

Cóc nước sần, ngóe sọc.

Occidozyga vittata (ANDERSSON, 1942)

Striped oriental frog, Forest puddle frog.

Cóc nước sọc, cóc nước nhỏ.

VI. Ranidae RAFINESQUE, 1814

Family Ranidae

Họ Ếch nhái

Hylarana erythraea (SCHLEGEL, 1837)

Green paddy frog, redeared frog, common greenback frog.

Chàng xanh.

14 15

Microhyla palmipes BOULENGER, 1897 * Micryletta inornata (BOULENGER, 1890)

СЕРИЯ

Физиология

Ếch nhẽo, ếch trơn, ếch nâu. Ếch gai sần, ếch núi, ếch da.

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Hylarana guentheri (BOULENGER, 1882)

Hylarana leptoglossa (COPE, 1868) *

Hylarana macrodactyla GṺNTHER, 1858

Guangdong frog, threestriped grass frog, longlegged grass frog.

Chàng hiu.

Hylarana milleti (SMITH, 1921)

Dalat frog, Millet’s frog.

Chàng mi lê.

Hylarana nigrovittata (BLYTH, 1856)

Black-striped frog.

Ếch suối.

Hylarana taipehensis (VAN DENBURGH, 1909)

Two-striped grass frog, taipei frog.

Chàng đài bắc.

Odorrana andersonii (BOULENGER, 1882)

Golden crossband frog, Anderson’s frog.

Chàng an đec son.

VII. Rhacophoridae HOFFMAN, 1932

Family Rhacophoridae

Họ Ếch cây

Polypedates leucomystax (GRAVENHORST, 1829)

Java whipping frog, Four-lined treefrog.

Ếch cây mép trắng.

Gymnophiona MṺLLER, 1831

Order Gymnophiona Bộ Không chân

Ichthyophiidae TAYLOR, 1968

Family Ichthyophiidae

Họ Ếch giun

Ichthyophis bannanicus YANG, 1984

Bannan caecilian.

Ếch giun, ếch giun ban na.

REPTILIA Laurenti, 1786

Class REPTILIA

Lớp Bò sát

Squamata OPPEL, 1811

Order Squamata

Bộ Có vẩy

Sauria MACARTNEY, 1803

Suborder Sauria

Phân bộ Thằn lằn

I. Agamidae GRAY, 1827

Family Agamidae

Họ Nhông

Physignathus cocincinus CUVIER, 1829

Indochinese water dragon, Green water dragon, Asian water dragon.

Rồng đất

Acanthosaura lepidogaster (CUVIER, 1829)

Scales-bellied tree lizard, Brown pricklenape.

Ô rô vẩy

Bronchocela smaragdina GṺNTHER, 1864

Emerald long-tailed agama, Guenther’s bloodsucker.

Nhông đuôi dài s - ma - ra

Calotes emma GRAY, 1845

Forest crested lizard, Emma forest lizard.

Nhông em - ma

Guenther’s Amoy frog, Guenther’s frog.

СЕРИЯ

Chẫu, chẫu chàng, chàng chuộc. Chàng lưỡi.

Физиология

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Calotes mystaceus DUMÉRIL & BIBRON, 1837

Moustached lizard, Blue-crested lizard, Indochinese forest lizard.

Nhông xám

Calotes versicolor (DAUDIN, 1802)

Garden fence lizard, Oriental garden lizard.

Nhông xanh

Draco indochinensis SMITH, 1928

Indochinese flying lizard, Indochinese gliding lizard.

Thằn lằn bay đông dương

Draco maculatus (GRAY, 1845)

Spotted flying lizard, Spotted gliding lizard.

Thằn lằn bay đốm

Draco volans LINNAEUS, 1758 **

Thằn lằn bay vạch

II. Gekkonidae GRAY, 1825

Family Gekkonidae

Họ Tắc kè

Gekko badenii SZCZERBAK & NEKRASOVA, 1994

Baden gecko

Tắc kè bà đen

Gekko gecko (LINNAEUS, 1758)

Gecko, Tokay.

Tắc kè, cắc kè

Hemidactylus bowringii (GRAY, 1845)

Oriental leaf-toed gecko, Bowring’s house gecko.

Thạch sùng bao rin

Hemidactylus frenatus SCHLEGEL, 1836

Common house gecko, Spiny-tailed house gecko.

Thạch sùng đuôi sần

Hemidactylus garnotii DUMÉRIL & BIBRON, 1836

Garnot’s house gecko, Indo-Pacific gecko.

Thạch sùng đuôi dẹp, thạch sùng ga - not

III. Lacertidae GRAY, 1825

Family Lacertidae

Họ Thằn lằn chính thức

Takydromus sexlineatus DAUDIN, 1802

Six-triped long-tailed grass lizard, asian grass lizard.

Liu điu chỉ, liu điu sáu vạch

IV. Scincidae GRAY, 1825

Family Scincidae

Họ Thằn lằn bóng

Eutropis longicaudata (HALLOWELL, 1856)

Long-tailed mabuya

Thằn lằn bóng đuôi dài

Eutropis macularia (BLYTH, 1853)

Bronze mabuya

Thằn lằn bóng đốm

Eutropis multifasciata (KUHL, 1820)

Lipinia vittigera (BOULENGER, 1894)

СЕРИЯ

East Indian brown mabuya, many-lined sun skink, commun sun skink. Striped tree skink, Banded lipinia, Common striped skink.

Физиология

Thằn lằn bóng hoa

Thằn lằn vạch

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Lygosoma bowringii (GṺNTHER, 1864)

Christmas island grassskink, Bowring’s supple skink.

Thằn lằn chân ngắn bao ring

Lygosoma quadrupes (LINNAEUS, 1766)

Short-limbed supple skink.

Thằn lằn chân ngắn thường

Sphenomorphus rufocaudatus DAREVSKY &NGUYEN, 1983

Red-tailed ground skink Thằn lằn phê - nô đuôi đỏ

V. Varanidae GRAY, 1827

Family Varanidae

Họ Kì đà

Varanus nebulosus (GRAY, 1831)

Clouded monitor

Kì đà vân, kì đà khô

Varanus salvator (LAURENTI, 1786)

Water monitor

Kì đà hoa, kì đà nước

Serpentes LINNAEUS, 1758

Suborder Serpentes

Phân bộ Rắn

I. Typhlopidae MERREM, 1820

Family Typhlopidae

Họ Rắn giun

Ramphotyphlops braminus (DAUDIN, 1803)

Common blind snake, Flowerpot snake.

Rắn giun thường, rắn giun

Typhlops diardii SCHLEGEL, 1839

Indochinese blind snake, Diard’s blind snake.

Rắn giun lớn, rắn giun di - ac

II. Cylindrophiidae FITZINGER, 1843

Family Cylindrophiidae

Họ Rắn hai đầu

Cylindrophis ruffus (LAURENTI, 1768)

Red-tailed pipe snake

Rắn trun, rắn hai đầu đỏ

III. Pythonidae FITZINGER, 1826

Family Pythonidae

Họ Trăn

Python molurus (LINNAEUS, 1758)

Asiatic rock python, Indian python, Burmese python.

Trăn đất, trăn mốc

Python reticulatus (SCHNEIDER, 1801)

Reticulated python

Trăn gấm

IV. Xenopeltidae BONAPARTE, 1845

Family Xenopeltidae

Họ Rắn mống

Xenopeltis unicolor REINWARDT, 1827

Sunbeam snake

Rắn mống, rắn hổ hành

V. Acrochordidae BONAPARTE, 1831

Family Acrochordidae

Họ Rắn rầm ri

Acrochordus javanicus HORNSTEDT, 1787

Java file snake, elephant-trunk snke.

Rắn rầm ri cóc, rầm ri gia - va

VI. Colubridae OPPEL, 1811

Family Colubridae

Họ Rắn nước

Calamaria pavimentata DUMÉRIL, BIBRON & DUMÉRIL, 1854

Collared reed snake

Rắn mai gầm lát

Ahaetulla nasuta (LACEPÈDE, 1789)

Long-nosed whip Rắn roi mõm nhọn snake, green vine snake.

СЕРИЯ

Физиология

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Ahaetulla prasina (REINHARDT, 1827)

Oriental whip snake, asian vine snake.

Rắn roi thường

Boiga cyanea (DUMÉRIL, BIBRON & DUMÉRIL, 1854)

Green cat snake

Rắn rào xanh

Boiga dendrophila (BOIE, 1827)

Boiga multomaculata (BOIE, 1827)

Mangrove snake, Goldringed cat snake Large-spotted cat snake, many-spotted cat snake.

Rắn rào cây Rắn rào đốm

Boiga siamensis NOOTPAND, 1971

Gray cat snake, eyed cat snake, ocellated cat snake

Chrysopelea ornata (SHAW, 1802)

Golden tree snake, Golden flying snake.

Rắn cườm

Coelognathus flavolineatus (SCHLEGEL, 1837)

Yellow-striped snake, Black copper rat nake, common malayan racer.

Rắn sọc vàng

Coelognathus radiatus (BOIE, 1827)

Copperhead racer, Radiated rat snake.

Rắn sọc dưa

Dendrelaphis pictus (GMELIN, 1789)

Common bronzeback, Painted bronzeback.

Rắn leo cây thường

Dryocalamus davisonii (BLANFORD, 1878)

Bridle snake, Blanford’s bridle snake.

Rắn dẻ

Gonyosoma oxycephalum (BOIE, 1827)

Red-tailed rat snake, red-tailed racer

Rắn lay, rắn đuôi đỏ

Lycodon capucinus BOIE in BOIE, 1827

Common wolf snake

Rắn khuyết mũ

Lycodon laoensis GṺNTHER, 1864

Laotian wolf snake, Indochinese wolf snake.

Rắn khuyết lào

Lycodon subcinctus BOIE, 1827

Malayan banded wolf snake

Rắn khuyết đai

Oligodon barroni (SMITH, 1916)

Barron’s kukri snake

Rắn khiếm ba - ron

Oligodon cinereus (GṺNTHER, 1864)

Ashy kukri snake, Guenther’s kukri snake.

Rắn khiếm xám

Oligodon fasciolatus (GṺNTHER, 1864)

Fasciolated kukri snake.

Rắn khiếm đuôi vòng

Oligodon formosanus (GṺNTHER, 1872)

Taiwan kukri snake

Rắn khiếm đài loan

Oligodon mouhoti (BOULENGER, 1914)

Mouhot’s kukri snake

Rắn khiếm mau- hô-ti

Oligodon ocellatus (MORICE, 1875)

Ocellated kukri snake.

Rắn khiếm vân đen

Oligodon taeniatus (GṺNTHER, 1861)

Striped kukri snake.

Rắn khiếm vạch

СЕРИЯ

Физиология

Rắn rào hoa

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Ptyas carinata (GṺNTHER, 1858)

Carinated rat snake, Keeled rat snake..

Rắn ráo gờ, rắn hổ mực gờ

Ptyas korros (SCHLEGEL, 1837)

Indochinese rat snake, Chinese rat snake.

Rắn ráo thường, rắn ráo, rắn lai

Ptyas mucosa (LINNAEUS, 1758)

Common rat snake, Oriental rat snake.

Rắn ráo trâu, rắn hổ trâu

Enhydris bocourti (JAN, 1865)

Bocourt’s water snake

Rắn bồng voi, rắn ri tượng, ri voi

Enhydris enhydris (SCHNEIDER, 1799)

Rainbow water snake

Rắn bông súng

Enhydris innominata (MORICE, 1875)

Mekong delta water snake

Rắn bồng không tên, rắn hổ lác

Enhydris plumbea (BOIE in BOIE, 1827)

Plumbeous water snake

Rắn bồng chì, rắn liu điu

Enhydris subtaeniata (BOURRET, 1934)

Mekong mud snake

Rắn bù lịch, Rắn bồng mê - kông

Erpeton tentaculatum LACEPÈDE, 1800

Tentaculed snake

Rắn râu

Homalopsis buccata (LINNAEUS, 1758)

Puff-faced water snake, masked water snake.

Rắn ri cá

Amphiesma stolatum (LINNAEUS, 1758)

Buff-striped keelback

Rắn sãi thường

Psammodynastes pulverulentus (BOIE, 1827)

Mock viper

Rắn hổ đất nâu

Rhabdophis subminiatus (SCHLEGEL, 1837)

Red-necked keelback

Rắn hoa cỏ nhỏ

Xenochrophis flavipunctatus (HALLOWELL, 1861)

Yellow-spotted keelback

Rắn nước, rắn nước đốm vàng

Pareas carinatus (BOIE, 1828)

Keeled slug snake

Rắn hổ mây gờ

Pareas laevis (BOIE in BOIE, 1827) ***

Slug snake.

Hổ mây trơn

Pareas margaritophorus (JAN, 1866)

White- spotted slug snake.

Hổ mây ngọc, rắn hổ mây đốm trắng

VII. Elapidae BOIE, 1827

Family Elapidae

Họ Rắn hổ

Bungarus candidus (LINNAEUS, 1758)

Blue krait.

Rắn cạp nia nam

Bungarus fasciatus (SCHNEIDER, 1801)

Banded krait.

Rắn cạp nong, rắn đen vàng

Naja siamensis LAURENTI, 1768

Indochinese spitting cobra, siamese cobra.

Rắn hổ mang xiêm

Ophiophagus hannah (CANTOR, 1836)

King cobra, hamadryad.

Rắn hổ chúa, Rắn hổ mang đen

Calliophis maculiceps (GṺNTHER, 1858)

Small-spotted coral snake.

Rắn vú nàng, Rắn lá khô đốm nhỏ

СЕРИЯ

Физиология

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

VIII. Viperidae OPPEL, 1811

Family Viperidae

Họ Rắn lục

Calloselasma rhodostoma (KUHL, 1824)

Malayan pitviper.

Rắn choàm quạp, rắn lục mã lai

Criptelytrops albolabris (GRAY, 1842)

White-lipped pitviper, White-lipped tree viper.

Rắn lục mép trắng

Tropidolaemus wagleri (BOIE, 1827)

Temple pitviper.

Rắn lục wag - lo

Testudines LINNAEUS, 1758

Order Testudines

Bộ Rùa

I. Geoemydidae THEOBALD, 1868

Family Geoemydidae

Họ Rùa thường

Cuora amboinensis (DAUDIN, 1801)

Malayan box turtle, Asian box turtle, Southeast Asian box turtle.

Rùa hộp lưng đen

Cyclemys dentata (GRAY, 1831)

Asian leaf turtle.

Rùa dứa

Cyclemys tcheponensis (BOURRET, 1939)

Stripe-necked leaf turtle.

Rùa đất sê - pôn

Heosemys annandalii (BOULENGER, 1903)

Yellow-headed temple turtle.

Rùa răng, càng đước

Heosemys grandis (GRAY, 1860)

Giant asian pond turtle.

Rùa đất lớn

Siebenrockiella crassicollis (GRAY, 1831)

Black marsh turtle, Siamese temple turtle.

Rùa cổ bự

II. Testudinidae GRAY, 1825

Family Testudinidae

Họ Rùa núi

Indotestudo elongata (BLYTH, 1853)

Elongated tortoise.

Rùa núi vàng, rùa gối, rùa voi

III. Trionychidae FITZINGER, 1826

Family Trionychidae

Họ Ba ba

Amyda cartilaginea (BODDAERT, 1770)

Asiatic softshell turtle, Black-rayed softshell turtle.

Cua đinh, ba ba nam bộ

Crocodylia GMÉLIN, 1789

Order Crocodylia

Bộ Cá sấu

Crocodylidae GRAY, 1825

Family Crocodylidae

Họ Cá sấu

Crocodylus siamensis SCHNEIDER, 1801

Siamese crocodile.

Cá sấu xiêm, cá sấu nước ngọt

Notes: Species*: according Bourret R. (1942) Species**: according Bourret R. (1943) Species***: according Bourret R. (1936)

СЕРИЯ

Физиология

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

Based on the data and the characteristic of the identification key, a relational database structure was built with 10 tables, in which, 6 tables contains information on different taxonomy levels, 3 tables contain features for identification

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at 3 taxonomy levels (family, genus, species), 1 table for the biological information of recorded species. Data in these table is connected by defined relationships as shown in the figure 1.

Figure 1: Relational database structure of the DAR For ease in accessing the data and identification of reptiles and amphibian in the studied region, a website was built and accessible at http://zoology.vietbiodata.net. Webpages are in Vietnamese, since most of users should be Vietnamese students or researchers. However, for more friendly, an English version has been developing and going to be available soon.

СЕРИЯ

Data stored in DAR can be mined in different ways. A list of 120 identified species is shown, in which, species are grouped based on taxonomy features. A link to the details of each corresponding species are also established as shown in Figure 2.

Физиология

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

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Figure 2: List of identified species Detail information of species consists of scientific name, Vietnamese name, biological features, location of distribution… as shown in Figure 3.

Figure 3: Detail information of an amphibia СЕРИЯ

Физиология

Hoa Pham Van, Quan Thai Ke

THE WEB - BASED TOOL FOR IDENTIFICATION OF AMPHIBIAN AND REP-TILES PRESENTED IN THREE WESTERN PROVINCES OF SOUTH - EASTERN REGION, VIETNAM

The identification tool is also included in the website. A Perl script was developed using the reported keys for identification of amphibian and reptiles. Every step, two options which are

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Сетевой научно-практический журнал

identification features are shown, one of them must be chosen for further identification. Pages with options would be repeated until a species is identified (Figure 4).

Figure 4: Steps in identification Conclusion The database of reptiles and amphibian in western provinces of South-easten region, Vietnam was established and can be accessed freely via Internet with webpages interface. It provides not only biological information about species in studied region but also the tool for identification of reptiles and amphibian. The tool is very user-friendly, can be used easily just by chosing the correct feature of the sample. In the near future, we will broaden the examined region as well as contribute to other groups to achieve a full database of reptiles and amphibian in Vietnam.

References: 1. Phạm Văn Hòa, 2005. Nghiên cứu khu hệ ếch nhái, bò sát các tỉnh phía Tây miền Đông Nam bộ (Bình Dương, Bình Phước, Tây Ninh). Biology doctoral thesis, Hue University, 153 pp. 2. Nguyen Van Sang, Ho Thu Cuc, Nguyen Quang Truong, 2009. Herpetofauna of Vietnam. Edition Chimaira, Frankfurt am Main, 768pp.

DATA ABOUT THE AUTHOR: Hoa Pham Van, Quan Thai Ke* Saigon University, Vietnam *Corresponding author: Quan Thai Ke. Email: quan.tk@cb.sgu.edu.vn

СЕРИЯ

Физиология

Сетевой научно-практический журнал СЕРИЯ

Физиология

А У Ч Н ЫЙ РЕЗУЛЬТАТ

УДК 576.322.2

Чернявских С.Д., Голдаева К.А., Дрыганова Л.А., Филиппенко Е.Г.

ФУКЦИОНАЛЬНЫЕ ОСОБЕННОСТИ СЕРДЕЧНОСОСУДИСТОЙ СИСТЕМЫ У ЮНОШЕЙ ПРИЗЫВНОГО ВОЗРАСТА

Аннотация

ходе исследования изучены функциональные особенности сердечнососудистой системы призывников в возрасте 18-21 года. Проведен анализ артериального, пуль-сового, среднего артериального давлений, а также произведена регистрация ЭКГ у юношей призывного возраста в состоянии относительного покоя и после нагрузки по 150 Вт. на велоэргонометре (ортостатическая проба с нагрузкой). Используя но-менклатуру зубцов комплекса PQRSТ, проанализированы и оценены ЭКГ-показатели II отведения: ЧСС, амплитуда зубцов P, Q, R, S и Т, длительность интер-валов P-Q, QRS и Q-T, рассчитаны показатели – интегративный (ИП) как отношение амплитуд зубцов Р и Т, систолический (СП) по формуле Фогельсона-Черногорова, определена должная электрическая систола по формуле Базетта. Установлено, что у испытуемых показатели, характеризующие состояние сердечнососудистой системы, как в покое, так и после функциональной пробы находились в пределах границ фи-зиологической нормы. После физической нагрузки по сравнению с состоянием по-коя в пределах нормальных величин увеличились значения показателей систоличе-ского и диастолического давления призывников. Анализ электрокардиограммы, проведенный после функциональной пробы, выявил снижение в пределах нормы показателей амплитуды зубцов Р, Q, R, S и Т, длительности интервалов P-Q, QRS и Q-T, должной электрической систолы и систолического показателя, а также повы-шение вагосимпатического индекса у юношей призывного возраста по сравнению с состоянием покоя.

лючевые слова: призывники; сердечнососудистая система; электрокардиограмма.

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G.

FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

Abstract

he study examines the functional characteristics of the cardiovascular system in 18-21 year old conscripts. The analysis of pulse, arterial and mean arterial pressure was performed. ECG was run for males of conscription age at the rest state and after the exercise load of 150 watts using Cardiac Stress Test (orthstatic load test). The data of II-lead was analysed using the standard method of PQRST peaks: Heart Rate, peak ampli-tude of P, Q, R, S, T, the length of P-Q, QRS, and Q-T intervals. The following parameters were measured: the integrative index (II) is the ratio of P and T peaks amplitude, the systolic index (SI) was measured with the Fogelson-Chernogorov formula. The proper electrical systole using Bazette formula was measured. It was found that the indicators of the test subjects’ cardiovascular system were within the physiological norm prior to and after the functional test. After the physical exercise, the systolic and diastolic pressure in conscripts increased compared to the resting state within the average values. The analysis of the ECG after the functional test revealed the decrease in the amplitude of P, Q, R, S, and T peaks, the length of P-Q, QRS, and Q-T intervals, proper electrical systole and systolic value, and the increase of vagosympathetic index in males of conscription age compared to the resting state.

ey words: : conscripts; cardiovascular system; electrocardiogram.

№2 2014

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G.

FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

Юношеский возраст – это период онтогенеза, в котором происходят разнообразные морфологические и функциональные изменения органов и систем, обуславливающие определенную уязвимость организма для развития ряда заболеваний [15]. На юношеском этапе жизни наблюдается значительная перестройка соотношений роста сердца и сосудов и в связи с этим возникает своеобразие условий кровообращения. Большую тревогу вызывает состояние здоровья юношей-призывников, так как в последние годы возврат юношей из армии по состоянию здоровья увеличился вдвое. Призывной возраст требует внимательного и дифференцированного подхода в дозировках физической нагрузки, с тем чтобы не вызвать явлений перегрузки, но одновременно с этим обеспечить и необходимую тренировку сердечнососудистой системы. Целью работы было изучение функциональных особенностей сердечнососудистой системы призывников. Материалы и методы исследования. Было обследовано четыре группы (по 25 человек) призывников. В первую группу вошли 18-летние юноши, во вторую – 19-летние, в третью – 20-летние, в четвертую – призывники в возрасте 21 года. Артериальное давление (АД) измеряли по Короткову в левой плечевой артерии, пульсовое давление рассматривали как разницу между систолическим и диастолическим артериальным давлением [4]. Среднее артериальное давление (АДср) определяли по формуле: АДср = (САД-ДАД)/3+ДАД, где САД – систолическое артериальное давление, ДАД – диастолическое артериальное давление.

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Регистрацию ЭКГ проводили в состоянии относительного покоя – лежа на спине после предварительного отдыха в течение 5 минут, а также на 6-й минуте после нагрузки по 150 Вт. на велоэргонометре (ортостатическая проба с нагрузкой) [7]. Запись ЭКГ проводили с помощью электрокардиографа «Аксион» при стандартном усилении 1 мВ=10 мм, скорости лентопротяжного механизма – 50 мм/с, в 12 общепринятых отведениях: в трех стандартных (I, II, III) однополюсных, усиленных от конечностей (аVR, аVL, аVF) и шести однополюсных усиленных грудных (V1-V6). Используя номенклатуру зубцов комплекса PQRSТ, анализировали и оценивали ЭКГ-показатели II отведения: ЧСС, амплитуду зубцов P, Q, R, S и Т, длительность интервалов P-Q, QRS и Q-T [14], рассчитывали показатели – интегративный (ИП) как отношение амплитуд зубцов Р и Т [12], систолический (СП) по формуле Фогельсона-Черногорова: (Q-T/R-R) ∙ 100%. Должную электрическую систолу определяли по формуле Базетта: Q-Tдолж = К√ R-R, где К – константа, равная для мужчин 0,37, для женщин – 0,39. Полученный цифровой материал был обработан статистически с использованием персонального компьютера [6]. При определении достоверности разницы между группами был использован критерий Стьюдента и таблицы Фишера-Снедекора по вычислению критерия достоверности. Результаты рассматривали как достоверные, начиная со значения p<0,05. Результаты исследования и их обсуждение. Показатели артериального давления у испытуемых всех групп соответствовали возрастной норме [10] (табл. 1). Таблица 1

Показатели артериального давления и ЧСС призывников

Table 1

The measures for arterial pressure and heart rate in conscripts Возраст, лет

Показатели, ед. изм.

АД систолическое, мм рт. ст. - до нагрузки - после нагрузки

118,0±3,88 149,0±4,33*

110,0±4,14 149,0±2,76*

124,0±2,49 166,5±2,11*

124,5±2,83 173,5±1,97*

СЕРИЯ

Физиология

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G.

FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

АД диастолическое, мм рт. ст. - до нагрузки - после нагрузки

77,0±2,13 86,0±2,21*

76,5±1,83 85,0±1,66*

80,0±1,22 87,0±1,53*

81,0±1,80 88,5±1,83*

Пульсовое давление, мм рт. ст. - до нагрузки - после нагрузки

41,0±2,33 63,0±4,72*

33,5±3,45 64,0±2,66*

45,0±1,67 78,5±2,36*

43,5±1,50 85,0±2,35*

Среднее артериальное давление, мм рт. ст. - до нагрузки -после нагрузки

90,6±2,62 106,96±2,14*

88,8±2,19 106,33±1,68*

97,6±1,93 118,49±2,35*

95,46±2,07 117,49±1,32*

ЧСС, уд./мин - до нагрузки - после нагрузки

82,7±1,48 154±3,11*

81,8±0,89 162±2,56*

82,3±0,79 161,2±3,05*

79,0±2,73 163,6±3,57*

При этом в пределах границ нормальных величин наблюдали незначительные сдвиги полученных данных. Так, после физической нагрузки у призывников 18, 19, 20 и 21 года систолическое давление было выше на 26, 35, 34 и 39%, диастолическое - на 12, 11, 9 и 9% соответственно по сравнению с показателями покоя. Аналогичные изменения были по показателям пульсового давления. Этот показатель был выше после функциональной пробы у испытуемых первой, второй, третьей и четвертой групп на 54, 91, 74 и 95% соответственно по сравнению с покоем. Показатели среднединамического давления, отражающие энергию непрерывного движения крови по сосудам, изменялись аналогично. Известно, что частота сердечных сокращений является одним из наиболее лабильных показателей гемодинамики. Данный показатель изменяется в процессе роста организма и зависит как от внешне средовых (температура окружающей среды, голод), так и от внутренних (поражение сердца, эндокринные расстройства, анемия и др.) факторов. В целом частота сокращений сердца как в покое, так и после физической нагрузки находились в пределах границ физиологической нормы у юношей всех групп, при этом наблюдали достоверное увеличение данного показателя после нагрузки у 18, 19, 20 и 21 летнем возСЕРИЯ

расте в сравнении с покоем соответственно на 86, 98, 96 и 107% (см. табл. 1). Как было сказано выше, основным инструментальным методом исследования сердечнососудистой системы является электрокардиография. По данным разных авторов, показатели ЭКГ (амплитуда зубцов, длительность интервалов) варьируют в широких пределах [7, 12, 14]. Исходя из этого, мы использовали для сравнения фоновые показатели (в покое) и показатели, полученные после физической нагрузки. В течение сердечного цикла записывали пять постоянных зубцов (P, Q, R, S и T), показатели которых представлены в таблице 2. Как видно из таблицы, все показатели зубцов после физической нагрузки снижаются практически в два раза. Зубец Р, характеризующий проведение возбуждения в предсердиях, после физической нагрузки снизился у 18, 19, 20 и 21-летних испытуемых на 48, 37, 50 и 50% соответственно по сравнению с состоянием покоя. Во всех группах этот зубец был положительным, что характеризует синусовый ритм. Зубец Q, характеризующий возбуждение межжелудочковой перегородки и верхушки сердца, у обследованных 18, 19, 20 и 21-летних призывников после нагрузки снизился на 44, 38, 43 и 47% соответственно.

Физиология

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G.

FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

Показатели амплитуды зубцов ЭКГ, мВ Показатели Р - до нагрузки - после нагрузки Q - до нагрузки - после нагрузки R - до нагрузки - после нагрузки

S - до нагрузки - после нагрузки T - до нагрузки - после нагрузки

The measures for ECG wave amplitude, mB Возраст, лет 18 19 20

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Таблица 2 Table 2 21

0,8±0,012 0,42±0,012*

0,7±0,009 0,44±0,016*

0,7±0,01 0,35±0,019*

0,9±0,007 0,45±0,013*

0,8±0,008 0,45±0,012*

0,6±0,008 0,37±0,015*

0,7±0,009 0,40±0,010*

0,9±0,008 0,42±0,006*

0,8±0,008 0,41±0,009*

0,6±0,110 0,37±0,015*

0,8±0,008 0,38±0,007*

1,0±0,009 0,42±0,010*

0,8±0,011 0,40±0,006*

0,6±0,008 0,37±0,011*

0,7±0,009 0,32±0,010*

0,9±0,007 0,42±0,008*

0,8±0,012 0,43±0,02*

0,7±0,006 0,40±0,009*

0,8±0,01 0,37±0,010*

1,0±0,009 0,43±0,001*

Зубец R, характеризующий возбуждение основной массы мускулатуры желудочков, кроме основания и субэпикардиального слоя, у испытуемых 18, 19, 20 и 21-летнего возраста снизился на 49, 38, 48 и 58% соответственно после физической нагрузки. При этом его значения, как в покое, так и после физической нагрузки у испытуемых обеих групп были в пределах границ нормальных величин. Амплитуда зубца S, отражающего состояние, когда возбуждены все отделы желудочков, кроме их основания, после физической нагрузки снизилась на 50, 38 ,54 и 53% у обследованных первой, второй, третьей и четвертой групп соответственно по сравнению с состоянием покоя. Известно, что физические нагрузки, сопровождаемые учащением сокращений сердца, могут способствовать возникновению гипоксии миокарда [11]. Для последней характерно снижение и инверсия зубцов Т. Более тяжелые формы гипоксии миокарда вызывают появление гигантских положительных зубцов Т. В возникновении этих изменений СЕРИЯ

существенное значение принадлежит особенностям распространения возбуждения от субэндокардиальных к субэпикардиальным слоям миокарда желудочков [12]. Согласно данным литературы [2, 11, 13], уплощение зубцов Т может также соответствовать метаболическим изменениям в самом миокарде как результат нарушения энергообеспечения и рассогласования активности центральных и автономных структур его регуляции. В нашем опыте во всех экспериментальных группах после физической нагрузки значения показателей зубца Т у испытуемых 18, 19, 20 и 21-летнего возраста уменьшились на 46, 43, 54 и 57% соответственно по сравнению с состоянием покоя, однако находились в пределах нормы, что может свидетельствовать об адаптации сердечнососудистой системы к гипоксии, а также стабилизации регуляторной функции. Кроме амплитуды зубцов нами была изучена длительность интервалов, результаты которой представлены в таблице 3.

Физиология

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G.

FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

АУЧНЫЙ РЕЗУЛЬТАТ

Сетевой научно-практический журнал

Показатели длительности интервалов ЭКГ, с

Таблица 3 Table 3

The measures for ECG intervals duration, s Показатели

Возраст, лет 18

PQ - до нагрузки - после нагрузки

0,08±0,002 0,07±0,002*

0,13±0,004 0,09±0,005*

0,11±0,003 0,09±0,004*

0,08±0,002 0,07±0,002*

QRS - до нагрузки - после нагрузки

0,06±0,002 0,05±0,001*

0,11±0,008 0,05±0,001*

0,05±0,006 0,05±0,003

0,07±0,004 0,07±0,002

QT - до нагрузки - после нагрузки

0,36±0,004 0,18±0,002*

0,28±0,005 0,15±0,006*

0,33±0,004 0,17±0,004*

0,31±0,006 0,18±0,003*

Как видно из таблицы, после физической нагрузки у призывников длительность интервалов P-Q в 18, 19, 20 и 21-летнем возрастах снизилась на 13, 31, 18 и 13% соответственно по сравнению с состоянием покоя. Известно, что соотношение длительности временных интервалов кардиоциклов отражает сопряженность вегетативных механизмов регуляции электрической активности сердца [2]. Снижение данного показателя после физической нагрузки по сравнению с состоянием покоя во временной структуре кардиоциклов доли интервалов Р-Q можно рассматривать как результат доминирующего влияния на электрическую активность сердца симпатического отдела ВНС [1, 3, 8, 9]. Длительность интервала QRS, характеризующего проведение возбуждения по рабочему миокарду желудочков, после функциональной пробы снизилась у 18 и 19-летних обследованных на 17 и 55% соответственно по сравнению с состоянием покоя. У испытуемых в возрасте 20 и 21 года после функциональной пробы значения данного показателя не изменились. После физической нагрузки у призывников длительность интервалов Q-Т в первой, второй, третьей и четвертой группах снизилась на 50 ,46, 48 и 42% соответственно в сравнении с покоем. При этом все показатели были в пределах нормы (см. табл. 3). СЕРИЯ

Длительность интервала Q-T отражает время, в течение которого желудочки находятся в электрически активном состоянии, и обозначается как электрическая систола. Продолжительность электрической систолы изменяется в зависимости от частоты сердечных сокращений. Установлена математическая зависимость между частотой сокращений сердца и длительностью интервала Q-T. Это так называемая должная электрическая систола. При нормальном состоянии сердца расхождения между фактической и должной систолой составляют не более 15% в ту или другую сторону. Полученные нами значения электрической фактической и должной систолы укладываются в данные параметры, что говорит о нормальном распределении волн возбуждения по сердечной мышце в обеих группах призывников как в покое, так и после функциональной пробы (табл. 4). После физической нагрузки у призывников в 18, 19, 20 и 21-летнем возрастах должная электрическая систола снизилась на 24, 10, 37 и 14%, фактическая – на 50, 46, 48 и 42% соответственно по сравнению с покоем. Распространение возбуждения по сердечной мышце характеризует не только длительность электрической систолы, но и так называемый систолический показатель, представляющий отношение длительности электрической систолы к продолжительно-

Физиология

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G.

FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

сти всего сердечного цикла. У испытуемых данный показатель снизился после нагрузки

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на 23, 48, 49 и 42% в первой, второй, третьей и четвертой группах соответственно.

Таблица 4 Показатели электрической систолы, систолического показателя и вагосимпатического индекса Table 4 The measures for electrical systole, systolic volume index and vagosympathetic index Показатели, ед. изм. Электрическая систола должная, с - до нагрузки - после нагрузки

Возраст, лет 18

0,33±0,002 0,25±0,002*

0,30±0,003 0,27±0,002*

0,38±0,003 0,24±0,003*

0,28±0,002 0,24±0,002*

Систолический показатель должный, % - до нагрузки - после нагрузки

0,44 0,57

0,55 0,48

0,66 0,57

0,65 0,56

Систолический показатель фактический, % - до нагрузки - после нагрузки

0,53 0,41

0,5 0,26

0,78 0,40

0,72 0,42

100 110,25

85,71 102,5

100 94,59

100 104,65

Вагосимпатический индекс - до нагрузки - после нагрузки

Интегративный показатель (Вагосимпатический индекс) характеризует отношение амплитуды зубца Р к зубцу Т. Увеличение данного показателя у призывников 18, 19 и 21 года после функциональной пробы на 10, 20 и 5% соответственно в сравнении с покоем свидетельствует о повышении тонуса симпатической нервной системы после физической нагрузки [12]. Заключение. Проведенные исследования подтвердили данные научной литературы, свидетельствующие о том, что экзогенная гипертермия способна повышать защитные свойства организма [21, 22]. Установлено, что при действии на организм интенсивной тепловой нагрузки в условиях развивающейся стресс-реакции происходит увеличение СЕРИЯ

площади спонтанной (на 39,6%) и стимулированной (на 30,7%) миграции лейкоцитов, фагоцитарной активности нейтрофилов (до 20,0±1,0%), при некотором повышении фагоцитарного индекса лейкоцитов. Кроме того под влиянием экзогенной гипертермии происходит не только стимуляция функциональных свойств белых клеток крови, обеспечивающих реализацию защитных реакций, но и активация механизмов, стабилизирующих геометрические показатели лейкоцитов. В условиях острого перегревания организма происходит компактизация структур клеток, сопровождающаяся снижением их объёма, пластичности и более экономным использованием ими мембранного резерва в среде с пониженной осмолярностью.

Физиология

Chernyavskikh S.D., Goldaeva Ch.A., Dryganova L.A., Filippenko E.G.

FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

Литература: 1. Васильев Н.В., Захаров Ю.М., Коляда Т.И. Система крови и неспецифическая резистентность в экстремальных климатических условиях. Новосибирск: Наука, 1992. 257с. 2. Киншт Д.Н., Киншт Н.В. Общая управляемая гипертермия: теория, практика, моделирование процессов. Владивосток: Дальнаука, 2006. 194 с. 3. Управляемая гипертермия / Баллюзек Ф.В., Баллюзек М.Ф., Виленский В.И., Горелов С.И., Жигалов С.А., Иванов А.А., Кузьмин С.Н., Определяков Г.А. СПб.: Невский диалект, 2001. 128 с. 4. Экспериментальные основы применения гипертермии в онкологии / Курпешев О.К., Лебедева Т.В., Светицкий П.В., Мардынский Ю.С., Чушкин Н.А. Ростов-на-Дону: Изд-во «НОК», 2005. 164 с. 5. Жаврид Э.А., Осинский С.П., Фрадкин С.З. Гипертермия и гипергликемия в онкологии. – Киев: Наукова думка, 1997. 256 с. 6. Горичева В.Д. Функциональные свойства и реактивность лейкоцитов крови в условиях гипертермии: Дис. … к.б.н. Ярославль: ЯГПУ им. К.Д. Ушинского, 2000. 133 с. 7. Moseley P.L. Heat shock proteins and heat adaptation of the whole organism // Journal of applied physiology. 1997. Vol.83. Issue 5. P. 14131417. 8. Козлов Н.Б. Гипертермия: биохимические основы патогенеза, профилактики, лечение. Воронеж: Изд-во Воронежского государственного университета, 1990. 104 с. 9. Пути практического использования интенсивного теплолечения (Второе сообщение) / Сувернев А.В., Иванов Г.В., Василевич И.В., Гальченко В.Н., Алейников Р.П., Новожилов С.Ю. Новосибирск: Академическое изд-во «Гео», 2009. 109 с. 10. Фёдорова М.З., Левин В.Н., Горичева В.Д. Функциональная активность и механические свойства лейкоцитов крови крыс при внешней тепловой нагрузке // Российский физиологический журнал им. И.М. Сеченова. 2000. № 12. С. 1624-1629.

References: 1. Vasiliev N.V., Zakharov Yu.M., Kolyada T.I. Blood System and Nonspecific Resistance under Extreme Climatic Conditions. Novosibirsk: Nauka, 1992. 257 p. 2. Kinsht D.N., Kinsht N.V. General Regulated Hyperthermia: Theory, Practice, Modeling of СЕРИЯ

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11. Медицинские лабораторные технологии / Под ред. Карпищенко А.И. – СПб.: Интермедика, 2002. 408 с. 12. Алексеев Н.А. Клинические аспекты лейкопений, нейтропений и функциональных нарушений нейтрофилов. СПб: Фолиант, 2002. 416с. 13. Дуглас С.Д., Куи П.Г. Исследование фагоцитоза в клинической практике. М.: Медицина, 1983. 112 с. 14. Зимин Ю.И., Редькин А.П. Угнетение нестимулированными лимфоцитами спонтанной миграции лейкоцитов под агаром // Иммунология. 1987. № 1. С. 71-73. 15. Фёдорова М.З., Левин В.Н. Метод комплексного исследования геометрии, площади поверхности, резервных возможностей мембраны и осморегуляции лейкоцитов крови // Клиническая лабораторная диагностика. 1997. №11. С. 44-46. 16. Выгодский М.Я. Справочник по элементарной математике. М.: АСТ: Астрель, 2006. 509 с. 17. Effect of acute heat stress on rat adrenal glands: a morphological and stereological study / Koko V., Djordjeviæ J., Cvijiæ G., Davidoviæ V. // The Journal of Experimental Biology. 2004. Vol.207. P.4225-4230. 18. Kluger M.J., Long N.C., Vander A.J. Stress-induced rise of body temperature in rats is the same in warm and cool environments // Physiology and behavior. 1990. Vol.47. No.4. P. 773-775. 19. Горизонтов П.Д., Белоусова О.И., Федотова М.И. Стресс и система крови. М.: Медицина, 1983. 240 с. 20. Селье Г. Стресс без дистресса. М.: Прогресс, 1979. 123 с. 21. The effect of mild whole-body hyperthermia on systemic levels of TNF-alpha, IL-1beta, and IL-6 in patients with ankylosing spondylitis / Tarner I.H., Müller-Ladner U., Uhlemann C., Lange U. // Clin. Rheumatol. 2009. Vol.28. P.397-402. 22. Whole body hyperthermia at 43.5-44°C: dreams or reality? / Suvernev A.V., Ivanov G.V., Efremov A.V., Tchervov R. // Hyperthermia in Cancer Treatment: A Primer. Medical Intelligence Unit. 2006. Section III. P.227-236. Processes. Vladivostok: Dal’nauka, 2006. 194 p. 3. Ballyuzek F.V., Ballyuzek M.F., Vilenskiy V.I., Gorelov S.I., Zhigalov S.A., Ivanov A.A., Kuz’min S.N., Opredelyakov G.A. Regulated hyperthermia. SPb.: Nevskiy dialekt, 2001. 128 p. 4. Kurpeshev O.K., Lebedeva T.V., Svetitskiy P.V., Mardynskiy Yu.S., Experimental Foun-

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FUNCTIONAL PECULIARITIES OF THE CARDIOVASCULAR SYSTEM IN YOUNG CONSCRIPT MALES

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dations of Hyperthermia Usage in Oncology. Rostov-na-Donu: Izd-vo «NOK», 2005. 164 p. 5. Zhavrid E.A., Osinskiy S.P., Fradkin S.Z. Hyperthermia and Hyperglycemia in Oncology]. Kiev: Naukova dumka, 1997. 256 p. 6. Goricheva V.D. Functional Properties and Reactivity of Blood Leucocytes under Hyperthermia Conditions: PhD Thesis in Biology. Yaroslavl’: YaGPU im. K.D. Ushinskogo, 2000. 133 p. 7. Moseley P.L. Heat shock proteins and heat adaptation of the whole organism. Journal of applied physiology. 1997. Vol.83. Issue 5. P. 1413-1417. 8. Kozlov N.B. Hyperthermia: Biochemical Basis of Pathogenesis, Prevention, treatment. Voronezh: Izd-vo Voronezhskogo gosudarstvennogo universiteta, 1990. 104 p. 9. Suvernev A.V., Ivanov G.V., Vasilevich I.V., Gal’chenko V.N., Aleynikov R.P., Novozhilov S.Yu. The ways of Practical Usage of Intense Warm-Treatment (The second report)]. Novosibirsk: Akademicheskoe izd-vo «Geo», 2009. 109 p. 10. Fedorova M.Z., Levin V.N., Goricheva V.D. Russian Physiological Journal. 2000. № 12. Pp. 1624-1629. 11. Medical Laboratory Technologies. Pod red. Karpishchenko A.I. SPb.: Intermedika, 2002. 408 p. 12. Alekseev N.A. Clinical Aspects of Leukocytopenia, Granulocytopenia and Neutrophils’ Functional Damages. SPb: Foliant, 2002. 416 p. 13. Duglas S.D., Kui P.G. Investigation of Phagocytosis in Clinical Practice. M.: Meditsina, 1983. 112 p.

14. Zimin Yu.I., Red’kin A.P. Immunology. 1987. № 1. Pp. 71-73. 15. Fedorova M.Z., Levin V.N. Clinical Laboratory Diagnostics. 1997. №11. Pp. 44-46. 16. Vygodskiy M.Ya. Elementary Mathematics Guide. M.: AST: Astrel’, 2006. 509 p. 17. Koko V., Djordjeviæ J., Cvijiæ G., Davidoviæ V. Effect of acute heat stress on rat adrenal glands: a morphological and stereological study. The Journal of Experimental Biology. 2004. Vol.207. Pp.4225-4230. 18. Kluger M.J., Long N.C., Vander A.J. Stress-induced rise of body temperature in rats is the same in warm and cool environments. Physiology and behavior. 1990. Vol.47. No.4. Pp. 773-775. 19. Gorizontov P.D., Belousova O.I., Fedotova M.I. Stress and blood system. M.: Meditsina, 1983. 240 p. 20. Sel’e G. Stress without distress. M.: Progress, 1979. 123 p. 21. Tarner I.H., Müller-Ladner U., Uhlemann C., Lange U. The effect of mild whole-body hyperthermia on systemic levels of TNF-alpha, IL-1beta, and IL-6 in patients with ankylosing spondylitis. Clin. Rheumatol. 2009. Vol.28. Pp.397-402. 22. Suvernev A.V., Ivanov G.V., Efremov A.V., Tchervov R. Whole body hyperthermia at 43.5-44°C: dreams or reality? Hyperthermia in Cancer Treatment: A Primer. Medical Intelligence Unit. 2006. Section III. Pp.227-236.

сведегия об авторах:

DATA ABOUT THE AUTHORs:

Чернявских Светлана Дмитриевна, доцент кафедры информатики, естественнонаучных дисциплин и методик преподавания, кандидат биологических наук, доцент Белгородский государственный национальный исследовательский университет, ул. Победы, 85, г. Белгород, 308015, Россия E-mail: Chernyavskikh@bsu.edu.ru Голдаева Кристина Александровна, студент Белгородский государственный национальный исследовательский университет, ул. Победы, 85, г. Белгород, 308015, Россия E-mail: 716215@bsu.edu.ru Дрыганова Лилия Александровна, студент Белгородский государственный национальный исследовательский университет, ул. Победы, 85, г. Белгород, 308015, Россия E-mail: 722247@bsu.edu.ru Филиппенко Елена Геннадьевна, студент Белгородский государственный национальный исследовательский университет, ул. Победы, 85, г. Белгород, 308015, Россия E-mail: 705776@bsu.edu.ru СЕРИЯ

Chernyavskikh Svetlana Dmitrievna PhD in Biology, Associate Professor Belgorod State National Research University 85 Pobedy St., Belgorod, 308015, Russia E-mail: Chernyavskikh@bsu.edu.ru Goldaeva Christina Aleksandrovna Student Belgorod State National Research University 85 Pobedy St., Belgorod, 308015, Russia E-mail: 716215@bsu.edu.ru Dryganova Liliya Aleksandrovna Student Belgorod State National Research University 85 Pobedy St., Belgorod, 308015, Russia E-mail: 722247@bsu.edu.ru Filippenko Elena Gennadiyevna Student Belgorod State National Research University 85 Pobedy St., Belgorod, 308015, Russia E-mail: 705776@bsu.edu.ru

Физиология

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УДК 591.111.1:595.76

Grebtsova E.A., Prisny A.A.

ENERGETIC AND MOTION ACTIVITY OF HEMOCYTES OF DICTYOPTERA REPRESENTATIVES

Abstract

he article is dedicated to results of the study, in the course of which motility of in-sects’ hemocytes, representatives of Dictyoptera order was investigated, while cells were incubated in various conditions. Morphological changes in blood plasma’s formed ele-ments were noticed in the course of fagocytal functions’ performance. It was shown that intensiveness of hemocytes’ fluorescence increases when cells with Rhodamine B colourant are incubated. Intensification of hemocytes’ fluorescence allows mentioning high energy needs for recognition of foreign objects and participation in phagocytosis.

ey words: hemocytes, motility, phagocytosis, rhodamine B, fluorescence, mitochondrial potential.

The complex of fluorescence colourants is used as optical indicator of membrane potential’s dynamics in cells and isolated organelles, which are too small to be tested via microelectrode potential technique. Insinuating cation fluorescent probes are selectively accumulated in live cells’ mitochondria. Mitochondrial-specific interaction of such molecules seems to depend on high transmembrane potential, supported by functioning mitochondria [1]. The usage of such potential-dependent probes is observed in the cells, participating in active movement. Such approach to analysis of mitochondrial membrane potential is important in the studies of energetic metabolism’s control and energetic requirements of certain biological functions at the cellular level [3]. Over the last years fluorescent colourants for measuring mitochondrial membrane potential (Δψm) have been frequently used for monitoring of changes of this important physiologic parameter, as well as for assessment of its correlation with cells’ ability to generate ATP by means of oxidative phosphorylation. Increase of fluorescence intensiveness directly depends on fluorescence elevation of mitochondrial potential [4]. Thus, Δψm is a key indicator of

normal or damaged cell state and indicator of their physiologic activity [5]. Hemocytes form the basis of insects’ cellmediated immunity. Plasmocytes, postulated homologues of mammals’ macrophages, as well as granulocytes of representatives of Dictyoptera order form 70% of locomotory subpopulation of circulating hemocytes [6]. They are responsible for tissue apoptosis occurring during metamorphosis, as well as for phagocytosis of bacteria. Up to date the mechanism of activation of plasma’s formed elements as a reaction to infection or developmental signals is still underinvestigated. Hemocytes of representatives of Dictyoptera order are motile ones; phagocytes are represented at all stages of their life circle and form cellular component of insects’ congenital immune system at postembryonic stage of life [7, 8, 9]. Granulocytes and plasmocytes are the most wide-spread types of cells. Thus, the main function of postembryonic hemocytes is elimination of intruding microorganisms and fragments of apoptotic cells. The objective of this work was to study motion activity and level of energetic metabolism of hemocytes of representatives of Dictyoptera order.

№2 2014

Grebtsova E.A., Prisny A.A.

ENERGETIC AND MOTION ACTIVITY OF HEMOCYTES OF DICTYOPTERA REPRESENTATIVES

Материалы и методы исследования. For meeting the objective set, studies of hemocytes of representatives of Dictyoptera order were implemented: Periplaneta americana, Shelfordella tartara, Gromphadorhina portentosa, Blaberus craniifer, Nauphoeta cinerea. The animals were kept in harden holding cages with coconut and sawdust soil. For successful animals’ breeding high level of humidity was kept. Animals were fed twice a week. Blood plasma was obtained per standard method [10]. Blood plasma obtained from the insect was incubated separately in physiological salt solution, as well as with addition of Saccharomyces cerevisiae cells in hypo- and hypertonic mediums. For assessment of energetic metabolism level in hemocytes, rhodamine B was used as a colourant. For preparation of working solution rhodamine B was diluted with 0,15М Phosphate buffered saline in the ratio 1:1500, and then diluted 10 times with 0.9 % NaCl solution. The cells that were incubated in various mediums were coloured and then were being observed for changes in fluorescence intenseness with the help of focal laser scanning microscope микроскопа Nikon Digital Eclipse Ti-E. Результаты исследования и их обсуждение. The most commonly encountered morphology of native hemocytes, incubated in physiological salt solution, which was denoted as “normal spreading” (NS), is a fully adherent cell with cellular membrane, having proportional bulging formations and cavities. This type is characterized by motionless sedentary behavior of these hemocytes. Two morphological classes, “non-polarized” (n/P) and “polarized” (P) are typical for hemocytes incubated in medium with addition of yeast cells. Both classes are fully adherent cells, demonstrating an extremely active cellular membrane, which forms bulges and cavities at various angles towards cellular midpoint area, forming ruffles. Significant difference between these two classes lies in the fact that polarized cells produce lamellipodia. Analysis of data, obtained from representatives of Dictyoptera order showed the absence of significant differences in peculiarities of motion activity and fluorescence intensiveness. With a view of data

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unification, in the course of discussion of the results the focus was set on hemocytes’ indices of Gromphadorhina portentosa. In natural conditions (incubation in physiological salt solution) 100 % of phagocytes were characterized by normal spreading state. At this, on the surface of granulocytes rhizopodia were distributed homogeneously and their length didn’t exceed 1.5 μm. Fluorescence intensiveness equaled 249±51 for granulocytes and 212±51 for plasmocytes. In hypotonic medium activity of both granulocytes and plasmocytes dropped. Upon termination of incubation period plasmocytes were found to be significantly spread. However, energy expenditures for keeping a membrane integral in conditions of reduced osmotic pressure were facilitated the increase of fluorescence intensiveness, which equaled 314±28 for plasmocytes and 387±40 for granulocytes. In conditions of increased osmotic pressure rhizopodium’s length reduced to 0.9 μm. The speed of plasmocydes’ spreading became significantly lower. Intensiveness of fluorescence was maximum, and it reached 554±48 for granulocytes and 420±36 for plasmocytes. No changes in motility compared with hemocytes incubated in physiological salt solution, was observed. On addition of yeast cells to hemocytes that had been kept in natural conditions, the percent of granulocytes with normal spreading equaled 34%, the number of polarized and non-polarized hemocytes reached 40% and 26%, respectively. There were no polarized hemocytes among plasmocytes, 92% of non-polarized hemocytes and 8% of hemocytes with normal spreading were observed. On the surface of granulocytes beside rhizopodia (the branchiness of which increased to 1.9 μm), long philopodia with a length of 3.5 μm appeared. Both types of pseudopodia were placed non-uniformly, often on one of the cellular poles, sideward to foreign agents. Intensiveness of phagocytes’’ fluorescence is similarly influenced by increased osmotic pressure and appearance of foreign objects in the medium. Besides, fluorescence intensiveness of phagocytes with normal spreading is higher in the medium, where yeast cells were added to, and is maximum for non-polar and polarized hemocytes (Table 1).

Физиология

Grebtsova E.A., Prisny A.A.

ENERGETIC AND MOTION ACTIVITY OF HEMOCYTES OF DICTYOPTERA REPRESENTATIVES

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Сетевой научно-практический журнал

Fluorescence intensiveness of hemocytes of various morphology Species of insect

Granulocytes n/P

Table 1

Plasmocytes NP n/P

Periplaneta americana

354±42

597±45

603±68

258±34

439±46

Shelfordella tartara Gromphadorhina portentosa Blaberus craniifer

351±39

588±51

599±47

251±42

431±39

346±60

581±67

596±82

245±48

425±54

342±69

573±60

591±77

240±30

419±25

Nauphoeta cinerea

337±43

569±37

587±46

238±26

411±38

Increasing of hemocytes’ fluorescence allows mentioning high energy needs for recognition of foreign objects and participation in phagocytosis. Заключение. Thus, it was stated that incubation of plasma formed elements in mediums different from physiological solution leads to increasing intensiveness of hemocytes’ fluorescence, which is evidence of increase of their energetic metabolism’s level. Study of hemocytes’ motion activity and level of energetic metabolism of representatives of Dictyoptera order allowed to elucidate that cellular reactions of studied five species of insects do not

have proved generic difference. In the course of the study three types of hemocytes’ morphological forms were determined, whose pseudopodia varied in type, character of location and motion activity. Two active morphological types of hemocytes – “non-polarized” and “polarized” were formed after foreign biological objects were placed to the medium. Incubation of plasma formed elements in mediums that contained foreign objects, as well as incubation of elements which differed from physiological salt solution, leads to increasing intensiveness of hemocytes’ fluorescence, which is evidence of increase of their energetic metabolism’s level.

References: 1. Johnson L.V., Walsh M.L., Bockus B.J., Lan Bo Chen. The Journal of Cell Biology, , 1981; 88: 526-535. 2. Perry S.W., Norman J.P., Barbieri J., Brown E.B., Gelbard H.A.,. Biotechniques 2011; 50(2): 98-115. 3. Grebtsova E.A., Universum: Chemistry and Biology 2014; 4(5): 11-19. 4. Lanot R., Zachary D., Holder F., Development Biology, 230: P. 243-257. 5. Russell C., Scaduto Jr., Grotyohann L.W. Biophysical Journal 1999;76: 469-477.

6. Grebtsova E.A., Prisny A.A. Novosibirsk 2012;P. 51-56. 7. Holz A., Bossinger B., Strasser T., Janning W, Klapper R. Development 2003; 130: 55-62. 8. Prisny A.A., Pigaleva T.A., Kulko S.V., Grebtsova E.A. Fundamental and Applied Science 2012; 3: 169-172. 9. Grebtsova E.A., Prisny A.A. new facts and hypotheses, 2014; 4: 91-93. 10. Prisny A.A. Belgorod National research University 2013; 116 p.

DATA ABOUT THE AUTHOR: Prisny Andrey Andreevich, PHD in biology, assistant professor Belgorod State National Research University 85, Pobedy St., Belgorod, 308015, Russia E-mail: Prisny@bsu.edu.ru

СЕРИЯ

Grebtsova Elena Alexandrovna post-graduate student Belgorod State National Research University 85, Pobedy St., Belgorod, 308015, Russia E-mail: shtirlitz009@mail.ru

Физиология