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A Practical Study on the Protocols of Invitro Fertilization

A Practical Study on the Protocols of Invitro Fertilization

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Categories:Types, Research, Science
Published by: Ahmad Mohammed Gumel on Apr 18, 2010
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11/06/2014

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1AHAMD SARKI GUMEL
Introduction/ Literature review:
Reproduction is the backbone of animal production. The possibility of enhancing reproductivepotentials through the use of certain biotechnological techniques has recently been explored bydifferent researchers(Da Ros, Maldera et al., 2008; George and Doe, 1989; Habsah, 2005; WanKhadijah, 2008). Cryopreservation is another important development facilitating conservationof valuable animal genetic, this captured the attention of many researchers(Kaneko, Yamamuraet al., 2006; Wan Khadijah, 2008). Several proteins such as OTP-1, bTP-1 and Interferonsecreted by the developing embryo have been identified and purified, these substances wereused as fertility promoters and markers of conception in different animal and human researchactivities(Srivistava A.K, 2005).It is now possible to reduce the generation interval by producing more number of offspringfrom a female within a short period of time using IVF and embryo transfer technology. Recentdevelopment in Öocytes maturation and invitro fertilization in farm animals have given a newdimension to animal production. Using such techniques it is possible to produce a large numberof embryos in the laboratory. Such embryos have a large demand in upstream areas of researchin embryo biotechnology. Brackett et al.(1982) produced the first IVF calf using ovulatedÖocytes, while Lu et al. (1987) reported birth of calf from total in vitro procedures ( in vitromaturation, fertilization development and culture of embryos). These reports generatedworldwide interest in science of invitro fertilization in livestock.In IVF matured Öocytes are coincubated with in vitro capacitated spermatozoa, which results infertilization and formation of zygote. The time of sperm-egg interaction, medium utilized,temperature and sperm co-incubation play important role in fertilization, and was studied bymany researchers(Brackett, 1982; George and Doe, 1989; Habsah, 2005; Heyers, Sousa et al.,2000; Kaneko et al., 2006; Koshimoto, Gamliel et al., 2000; Miao, Shi et al., 2007; Ozgunen,Erdogan et al., 2001; Zavos, Correa et al., 1994). It was observed that 3 hours after the releaseof first polar body is the best time for coincubation. Early as well as delayed incubation leads todestruction of Öocytes, failure of pronuclei fusion and cleavage(Srivistava A.K, 2005).Major steps involve in invitro fertilization are collection of Öocytes, in vitro maturation of Öocytes, in vitrocapacitation of spermatozoa, in vitro fertilization and invitro embroygenicdevelopment.Success of IVF in any species depends primarily on recovery of good quality Öocytes. OvulatedÖocytes can be collected by laparoscopy; invivo collection of Öocytes has been expensive timeconsuming and not practically possible(Srivistava A.K, 2005). Ovaries obtained from slaughterhouse may be an alternative, easy and cost effective method for collection Öocytes; however,in Malaysia, this may not be applicable, as most of the mature and reproductive female
 
2AHAMD SARKI GUMEL
animalsare kept in the farm for production, those taken to slaughter house are either too oldnot in their prime reproductive age or are too young for their reproductive system to producethe required Öocytes to harvested. Once taken from the slaughter house ovaries are brought innormal saline at 35
o
C-38
o
C and Öocytes are harvested immediately. The ovaries can be kept at20-24
o
C for 8-10 hours after the slaughter without the reduction in fertilizing ability of Öocytesand their subsequent development(Srivistava A.K, 2005).Another issue beside Öocytes recovery is sperm capacitation, which is the process by whichspermatozoa undergo certain morphological and biochemical changes before taking part in theprocess of fertilization. Capacitation occurs as a result of decreased negative surface charges,loss of membrane cholesterol and an influx of calcium between the plasma and outeracrosomal membrane. In this practical study capacitation was done by incubating the collectedsperm in TYH/BSA medium in 5% CO
2
incubator for 1 hour at 37
o
CEmbryo Transfer(ET) is one of the techniques of reproductive biotechnology where embryosflushed from donor females are transferred to the recipients, which serve as foster mother fortheir development throughout the period of pregnancy. W. Heape in 1890 made the firstsuccessful embryo transfer in rabbit(Srivistava A.K, 2005). However systematic research onembryo transfer wasnt started till after the birth ofWilmuts calf; first calf embryo transfer outof frozen embryo(Rowson, 1973). The production of embryos can be increase manifold throughsuperovulation, not only does it influence the embryo production, but it help to exploits the useof germ pool of ovarian reserve to the maximum level. Hormones like
Pregnantmares
 
serum gonadotrophin (PMSG), human chorionic gonadotropin (hCG), humanmenopausal gonadotropin (hMG), follicle stimulating hormone (FSH) and equinechronic gonadotrophin (eCG) are among the most commonly used superovulatoryhormones.
In general, the technology of Artificial insemination has the potential application inconservation of germplasm line, disease control and salvaging the reproductive problems.
Obj
ective:
The aim of this practical study was:
y
 
To be able to demonstrate and apply what we have learnt in class lecture in techniquesof IVF
 
y
 
To learn how to use the equipment in dissecting both male and female mouse andrecover the spermatozoa and Oocytes respectively.
 
 
3AHAMD SARKI GUMEL
y
 
To observe and learn the different reproductive tract of the mouse putting emphasis ontheir size and appearance when ovulated or the regions that have the sperm and theirposition for applying the knowledge in future IVF work.
 
y
 
To learn the techniques employed in storing the recovered sperm and Oocytes usingdifferent invitro culture media and the techniques of using CO2 incubator
 
y
 
To learn how to make invitro fertilization and incubate the fertilized oocytes using theCO2 incubator.
 
y
 
To learn the technique employed in embryo recovery from copulated female mouse
 
y
 
To observe any short comings during the process and make possible suggestions.
 Materials and Methods:
Animal Samples: laboratory Male mouse and Superovulated female mice (12 hourspostovulation) sacrificed humanely
Medium:
TYH (Toyodo Yokoyama Hoshi) medium, WM (Whitten Medium), HWM (Hepes bufferedWhitten medium),BSA (Bovine serum Albumen)
Equipment:
4, 5 forceps, 4 dissecting scissors, 6 dissecting scissors, dissecting board, aluminum foil,gauze, 4-well culture dish, glass micropipette, hand gloves, stereomicroscope
Reagents:
70% ethanol
Methodology:1.0 Sperm Recovery:
We were provided by a matured male mouse thatspent some period isolated from females .We killed theanimal by cervical dislocation using edge of a rubber cup in the laboratory sink.The cup-edge was placed at the base of the skull, little pressure was applied while twisting theedge on the neck and pulling the tail backward, at start the mouse was struggling, then afterabout two minutes we observed the mouse to be like in a quite sleep, by then noticed that ithas been killed.

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