ELECTRONIC AUTOMATIC TEMPERATURE CONTROL OF

CRYOPRESERVATION FOR ARTIFICIAL INSEMINATION (PROTOTYPE)

SANJOY KUMAR DEBNATH

A project report submitted in partial fulfilment of the

requirements for the award of the degree of

Master of Engineering Electrical- Mechatronics & Automatic Control

Faculty of Electrical Engineering

Universiti Teknologi Malaysia

JUNE 2013

iii

To my Father, Mother

&

Teachers

iv

ACKNOWLEDGEMENT

First and foremost, I would like to express heartfelt gratitude to my supervisor

Dr. Shafishuhaza Shalan for her constant support during my study at UTM. She

inspired me greatly to work in this project. Her willingness to motivate me contributed

tremendously to my project. I have learnt a lot from him and I am fortunate to have

her as my mentor and supervisor.

Besides, I would like to thank the authority of Universiti Teknologi Malaysia

(UTM) for providing me with a good environment and facilities such as Computer

laboratory to complete this project with software which I needed during the study.

v

ABSTRACT

The scientist and researchers are facing problems in genetically stabilizing

living cells. The living cells can be stabilized by keeping them in cryogenic

temperatures. Stabilizing cells at cryogenic temperatures is called cryopreservation, an

applied aspect of cryobiology, or the study of life at low temperatures. Advances in

cryopreservation technology have led to methods that allow low-temperature

maintenance of a variety of cell types. Techniques are available for the preservation of

microorganisms, isolated tissue cells, small multi cellular organisms, and even more

complex organisms such as embryos. The freezing process involves complex

phenomena which, even after decades of research, are not fully understood. The aim

of this project to make prototype of a cryopreservation system, so, it can preserve the

sperm or any other biological material. For the aim of cryopreservation, a circuit

board with a microcontroller, thermocouple controller, and a voltage driver for valve

are constructed. Two sensors are used, a K-types thermocouple and Water level

detector-brick sensor, which send data to the microcontroller. After the

microcontroller receives the data, the data of temperature and level are compared to

control the valve. The cryopreservation system is modeled in AutoCAD and the

temperature control circuit is programmed in MPLAB. An adjustable plastic box

which is non-conductive and resistant to heat is used in the fabricated system. It has a

metal box inside to reserve sperm tubes. An electrical solenoid valve is used for

supplying necessary liquid. The total cryopreservation is activated by sensing physical

value by the sensors which are converted by thermocouple amplifier in understandable

form for microcontroller. The data are then processed, compared and the valve is then

operated accordingly by the microcontroller. The fabricated system is tested at

different temperature conditions and it is found that there is very less error. The

system functions very effective and is able to control the temperature of the system to

preserve the sperm.

vi

ABSTRAK

Para saintis dan penyelidik menghadapi masalah dalam genetik menstabilkan

sel-sel hidup. Sel-sel hidup boleh stabil dengan menyimpannya dalam suhu kriogenik.

Penstabilan sel-sel pada suhu kriogenik dipanggil krioawetan, satu aspek gunaan

cryobiology, atau kajian hidup pada suhu rendah. Kemajuan dalam teknologi

krioawetan telah membawa kepada kaedah yang membolehkan penyelenggaraan

rendah-suhu pelbagai jenis sel. Teknik yang disediakan untuk pemeliharaan

mikroorganisma, sel-sel tisu terpencil, organisma kecil yang berbilang sel, dan

organisma yang lebih kompleks seperti embrio. Proses pembekuan melibatkan

fenomena yang kompleks tidak difahami sepenuhunya, walaupun beberapa dekad

penyelidikan. Tujuan projek ini untuk membuat prototaip sistem krioawetan, jadi, ia

boleh memelihara sperma atau apa-apa bahan biologi lain. Bagi tujuan krioawetan,

papan litar dengan pengawal mikro, pengawal suhu dan pemandu voltan untuk injap

dibina. Dua sensor digunakan, suhu K-jenis dan paras air sensor pengesan-bata, yang

menghantar data ke mikropengawal. Selepas mikropengawal menerima data, data

suhu dan tahap dibandingkan untuk mengawal injap. Sistem krioawetan adalah

model dalam AutoCAD dan litar kawalan suhu diprogramkan dalam MPLAB. Sebuah

kotak plastik laras yang bukan konduktif dan tahan haba yang digunakan dalam

sistem yang direka. Ia mempunyai kotak logam dalam untuk tiub sperma rizab. Injap

solenoid elektrik digunakan untuk membekalkan cecair yang diperlukan. Jumlah

krioawetan diaktifkan dengan mengesan nilai fizikal oleh sensor yang diubah oleh

penguat suhu dalam bentuk mudah difahami bagi mikropengawal. Data-data ini

kemudiannya diproses, dibandingkan serta injap itu dikendalikan sewajarnya oleh

pengawal mikro. Sistem fabrikasi diuji pada keadaan suhu yang berbeza dan ia

didapati bahawa terdapat ralat yang sangat minima. Fungsi sistem ini sangat efektif

dan mampu untuk mengawal suhu sistem untuk memelihara sperma.

vii

TABLE OF CONTESTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF APPENDICES xiii

1 INTRODUCTION 1

1.1 Background 1

1.2 Problem Statement 3

1.3 National and international status about AI 3

1.4 Objectives 4

1.5 Scope of work: 5

viii

2 LITERATURE REVIEW 6

2.1 Introduction 6

2.2 LN2 storage phases 6

2.3 Temperature Control Flochart 8

2.4 Temperature Control Flochart 9

2.5 Reasons for co-locating electronics 10

with sensors in a cold or hot environment include

3 RESEARCH METHODOLOGY 13

3.1 General View 13

3.2 Project work Methodology 14

3.3 Sensor works 15

3.4 The Design Analysis 16

3.4.1 Cool box 16

3.4.2 The metal box

3.4.3 The gap between cool box and metal box 18

3.4.4 Supporting water Tank 18

3.4.5 Solenoid Valve 19

3.5 Component Required for Temperature Control 19

3.6 Temperature control circuit Design 21

3.7 Type K Thermocouple 22

3.7.1 Linearity check & testing the temperature sensor 23

3.8 Thermocouple Amplifier SN-6675 with MAX6675 24

3.9 Water Brick Sensor 27

3.10 Testing with Water Detecting Sensor 29

3.11 Electrical Solenoid Valve 29

3.12 MD10C, Motor Driver 32

3.12.1 General Information of MD10C 32

3.12.2 Product Specification & Limitations 33

3.12.3 Board Layout 34

4 EXPERIMENT, RESULT AND DISCUSSION 37

4.1 Display and controller test 37

4.2 Functionality testing of the system and the results 38

ix

5 CONCLUSION 40

5.1 Conclusion 40

5.2 Recommandation 41

REFERENCES 41

Appendices A-B 44-49

x

LIST OF TABLES

TABLE NO. TITLE PAGE

1.0 Data of AI cases in different years in Malaysia 03

3.1 List of component required for Temperature Control 18

3.2 Temperature in thermometer corresponding to 23

voltage in digital meter

3.3 Comparison of different types of Valve 30

3.4 Specification of MD10C 32

3.5 Pin description of MD10C 33

3.6 Pin description of MD10C 34

3.7 Truth table of control logic 34

xi

LIST OF FIGURES

TABLE NO. TITLE PAGE

1.1 Graphical representation of AI in Malaysia 04

2.1 Vapor phase storage and liquid phase storage 07

2.2 Flow Chart showing the sensors and 09

controller combine works

2.3 Semen Handling 10

3.1 Project Work Methodology 13

3.2 A smart sensor architecture that 14

the same idea used in this project

3.3 Sensor‟s sensing flow diagram 14

3.4 Top view of Auto CAD drawing design of the system 15

3.5 Side view of Auto CAD drawing, design of the system 15

3.5.1 Top view of real system showing the sensors inside 16

3.5.2 Top view of real system 17

3.6 Schematic diagram of interfacing with 20

microcontroller an thermocouple.

3.7 36" high temperature Type-K Thermocouple 21

with glass braids insulation

3.8 Linearity check of Thermocouple 22

xii

3.9 Graphical representation of linearity of thermocouple 24

3.10 Thermocouple amplifier SN-6675 25

3.11 Interfacing of MAX6675 25

3.12 Real circuit connection using the thermocouple, 26

Thermo couple amplifier and with microcontroller

26

3.13 A Brick Water detector Sensor 27

3.14 3.14A When Out of water less voltage drop 28

3.14B Inside water greter voltage drop 28

3.14 C When Out of water less voltage drop 29

3.14D Inside water greter voltage drop 29

3.15 Dimension of MD10C 32

3.16 Board layout of MD10C 33

3.17 Connection between MD10C and SK40C 35

3.18 Connection between MD10C and SK40C 35

4.1 Test of display in microcontroller display 36

4.2 The integrated system during the testing 37

xiii

LIST OF APPENDICES

APPENDIX TITLE PAGE

A program wrote in „C‟ language and 44

used for test the display with MPLAB.

B This Program for temperature control in 46

MPLAB main function written in „C‟ language:

1

CHAPTER 1

INTRODUCTION

1.1 Back ground:

Artificial insemination (AI) – is one of the most imperative techniques ever

devised for the genetic enhancement of farm animals. It is used extensively as a tool

for livestock breeding and management programs. AI is a procedure by which

sperms are collected from the male, processed, stored and artificially introduced into

the female reproductive tract for the purpose of conception.

A male animal produces millions of sperms daily. Theoretically, it can

inseminate females regularly and produce several off springs. Artificial insemination

is used instead of natural mating for reproduction purposes. This is when a male

animal, for example, a bull, is kept with a herd of cows and ‗covers‘ (copulates with)

them when they are ready to mate so the bull‘s semen fertilizes the cow‘s eggs to

produce calves. Fertilization can take place away from the bull and the two animals

do not even meet. Although AI (in the form of intrauterine insemination) is not

frequently used in human patients, it is the most commonly used method of breeding

food production animals in developed countries, with more than 90% pigs and almost

the same proportion of dairy cattle bred by this method in the European Union and

North America [1].

In the actual procedure used, semen is obtained from a male animal and, after

being diluted, is deep-frozen, after which it can be stored for long periods of time

without losing its fertility. For use, the semen is thawed and then introduced into the

genital tract of a female animal.

Cryobiology is the study of the effects of extremely low temperatures on

biological systems, such as cells or organisms. Cryopreservation—an applied aspect

2

of cryobiology has resulted in methods that permit low temperature maintenance of a

diversity of cells.

The objective of cryopreservation is to minimize damage to biological

materials, including tissues, mammalian cells, bacteria, fungi, plant cells and viruses,

during low temperature freezing and storage. Cryopreservation provides a

continuous source of tissues and genetically stable living cells for a variety of

purposes, including research and biomedical processes.

A basic principle of cryobiology is that the extent of freezing damage

depends on the amount of free water in the system and the ability of that water to

crystallize during freezing. Water is the major component of all living cells and must

be present for chemical reactions to occur within a cell. During freezing, most of the

water changes to ice, and cellular metabolism ceases. However, obtaining

reproducible results for more complex tissues, such as heart valves or engineered

tissue constructs, or more sensitive cell types, requires an understanding of the major

variables involved in tissue cryopreservation [1].

The first successful experiment with artificial insemination in animals was

performed by an Italian physiologist Lazzaro Spallanzani in 1780. He developed a

technique for artificial insemination in dogs. This approach was distinguished

commercially in 1930 in Russia and the subsequent development of methods for the

cryopreservation was developed.

Cryopreservation is a process where cells or whole tissues are preserved by

cooling to sub-zero temperatures, typically 77 K (= −196 °C, the boiling point of

liquid nitrogen) of semen led to the wide spread use of AI in animals[4].

There are many advantages to artificial insemination (AI) in domesticated

and zoo animals. They are smaller chance of injury to either partner during the

mating process, X Preface less stress to the female, who is often the one transported

to and from the home of the male, the system of reproduction is perfect, including

artificial insemination. Examples of wild animals that have been successfully

impregnated through artificial insemination include big cats (e.g., the tiger, the puma,

the cheetah, and the clouded leopard etc.) [9].

3

1.2 Problem Statement

Cryopreservation is a process where cells or whole tissues are preserved by

cooling to sub-zero temperatures, typically 77 K (= −196 °C, the boiling point of

liquid nitrogen). The control strategy of temperature until now is done manually and

the maintenance is done by rule of thumb or experience. Hence automatic

temperature control system is required which can give significant compensation in

production of animal or person. It will also be economical and does not create losses

to farm industries.

The motivation for this project is to have Cryopreservation which can control

temperature automatically and can preserve sperm for a long time. The

transportation will also be easy which can minimize the risk of damage to the semen

and can increase the farm production.

1.3 National and international status about AI

As per Food and Agriculture Organization of United Nations (FAO), AI

coverage in developed countries in 1995 for dairy cattle was 64% and 7.8% for beef

cattle compared to developed countries, AI application in Asian countries is very

low. In Chi na it was 12.6% 1993 and in Indonesia it was 17.4% in 2000 and 23.1%

in 2002. For Malaysian scenario, the AI coverage was 4.3% in 2002 and it remained

same till 2009, which is quite similar for several years and is extremely low rate

measure compared to Indonesia [2].

4

Table 1.0: Data of AI cases in different years in Malaysia [2].

Year 1981 1982 1983 1984 1985 1986 1987 1988

Cases 13,357 15,466 19,282 20,532 19,930 22,072 19,533 22,864

Year 1989 1990 1991 1992 1993 1994 1995 1996

Cases 22,787 25,864 19,145 23,372 24,862 22,393 15,972 22,325

Year 1997 1998 1999 2000 2001 2002 2003 2004

Cases 19,243 16,650 15,845 17,527 16,208 18,276 16,867 15,810

Year 2005 2006 2008 2008 2009

Cases 15,676 18,369 19,021 19,752 18,599

Fig 1.1: Graphical representation of AI in Malaysia [2]

0

5,000

10,000

15,000

20,000

25,000

30,000

19

81

19

82

19

83

19

84

19

85

19

86

19

87

19

88

19

89

19

90

19

91

19

92

19

93

19

94

19

95

19

96

19

97

19

98

19

99

20

00

20

01

20

02

20

03

20

04

20

05

20

06

20

07

20

08

20

09

Number of Aritficial Insermination cases in Peninsula Malaysia(1981-2009)

Number of Aritficial Insermination cases in Peninsula Malaysia(1981-2009)

5

1.4 Objectives:

This project aims to achieve the following objectives;

To design and fabricate an automatic temperature control cryopreservation

device for sperm storage which can maintain the quality of sperm and increase the

animal production with less labor cost.

1.5 Scope of the project:

The Project is developed for automatic control of the temperature of the

cryopreservation system. The following are the scope of the project;

The artificial insemination and cryopreservation system is to be studied to

appreciate the significance of cryopreservation system in AI. This study will identify

the quality assurance for sperm or any other similar biological material and the

importance of freezing rate for controlling the temperature. Design a

Cryopreservation tank using a computer aided design tool (CAD). Auto CAD 2010

is to be used to model the plastic cool box with metal box inside which is a good

conductor of temperature.

Design a temperature control circuit for controlling the system with glass

braid K type with range of -270 to +1372 C thermocouple sensor, a thermocouple

amplifier SN-6675 for amplifying the weak voltage and to send the amplified signals

to the microcontroller.

Water detects Brick sensor is to be used which will detect the water level.

An electrical solenoid valve AQT15S made up by plastic material and operated by

DC12V or DC24 V or 220 AC is to be used. This valve in the system is to control

the flow of liquid or water inside the tank. The one end of the valve attached to

preserver and other end is attached to the tank. It is driven by a motor driver

MD10C. SK40C model microcontroller system with PIC16F877A and PIC Kit 2

6

V2.61 is to be used to control the entire system. MPLAB IDE V8.46 is to be used

for Programming the microcontroller. It has a display unit to display the temperature

and hence monitoring will be easier. The micro controller programming is to be

written in a high level language ‗C‘.

42

REFERENCES:

[1] Baril G, Leboeuf B, Saumande J. Synchronization of estrus in goats: the

relationship between time of occurrence of estrus andfertility following

artificial insemination. Theriogenology1993; 40:621–8.

[2] Raymond A.K. and Saifulliazam A.K.‖The cattle artificial insemination

industry in Malaysia‖. Department of veterinary service.Wisma Tani,Presint

462630 Putrajaya,,Malaysia.

[3] Thermo Fisher Scientific Inc( 2011) .cryopreservation manual.[Broucher].Dr.

Kelvin

[4] Milad Manafi (june 2011),Artificial Insemination in Farm Animals (2nd

edition) ;InTech Janeza Trdine 9, 51000 Rijeka, Croatia

[5] Dorado J, Rodrı´guez I, Hidalgo M. Cryopreservation of goat spermatozoa:

comparison of two freezing extenders base onpost-thaw sperm quality and

fertility rates after artificial insemination.Theriogenology 2007; 68:168–77.

[6] Batista M, Alamo D, Gonza´lez F, Cruz MG, Gracia A. Influence of the

freezing technique (nitrogen liquid vs ultrafreezer of 152 8C) and male-to-

male variation over the semen quality in Canarian Mastiff breed dogs. Reprod

Domest Anim 2006;41:423–8.

[7] Mazur, P. ―Fundamental cryobiology and the preservationof organs by

freezing.‖ In Organ Preservation for transplantation. (Eds. A. M. Karow and

D. E.Pegg), Marcel Dekker, New York, pp. 143-175, 1981.

[8] ―The effect of liquid nitrogen submersion on cryopreserved human heart

valves.‖ Cryobiology 27: 605-614, 1990 05-614, 1990.

[9] Production and storage of goat semen for artificial insemination by B.

Leboeuf a,) B. Restall b, S. Salamon c.

[10] Asch, R.H., J. Balmaceda, and C.J. Pauerstein (1978) A new approach to

studies of sperm transport in the rabbit by isotope labeling. Fertil. Steril.,

29.252.

[11] G. Battistoni, D. V. Camin, N. Fedyakin, G. Pessina,M. Sironi, ―Cryogenic

performance of monolithic MESFET preamplifiers for LAr calorimetry,‖

43

Nuclear Instruments & Methods in Physics Research A, vol.A395, no.1,

pp.134-140, 1 Aug. 1997.

[12] NASA- Glenn Research Center, Extreme Electronics, America, December 8,

2012

[13] NASA-Extreme Cryopreservation Electronics, America, November 23, 2012

[15] Adam, M, J. F. Hu, P. Lange, and L. Wolfinbarger.―The effect of liquid

nitrogen submersion on cryopreserved human heart valves.‖ Cryobiology 27:

6Adam, M, J. F. Hu, P. Lange, and L. Wolfinbarger.

[16] Written by Frank P. Simione, M.S. of the American Type Culture Collection

(ATCC) in cooperation with Nalge Nunc International Corp.©Nalge Nunc

International Corp. 1998

[17] Advance Instruments and Measurements, class note. [MEM 1753] (2012) .

Professor Dr. Ruzairi Abdul Rahim.UTM.Malaysia.

[18] .Cytron Technologies. HEAD to TOE, Product User‘s Manual – MD10C,

Trade brochure. 2013.

[19] National Instrumentation, Sensor Terminology, Malaysia; retrieve- May 17,

2013

[20] Cytron Technologies, Sensor works and projects, Malaysia, December 2012.