effect of sperm and oocytes factors on effect of sperm and oocyte factors on intracytoplasmic sperm

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  • EFFECT OF SPERM AND OOCYTE FACTORS ON

    INTRACYTOPLASMIC SPERM INJECTION (ICSI)

    PERFORMANCE IN MICE AND GOATS

    NOR FARIZAH BINTI ABDUL HAMID

    FACULTY OF SCIENCE

    UNIVERSITY OF MALAYA

    KUALA LUMPUR

    2012

  • EFFECT OF SPERM AND OOCYTE FACTORS ON

    INTRACYTOPLASMIC SPERM INJECTION (ICSI)

    PERFORMANCE IN MICE AND GOATS

    NOR FARIZAH BINTI ABDUL HAMID

    DISSERTATION SUBMITTED IN FULFILLMENT OF

    THE REQUIREMENT FOR THE DEGREE OF

    MASTER OF SCIENCE

    INSTITUTE OF BIOLOGICAL SCIENCES

    FACULTY OF SCIENCE

    UNIVERSITY OF MALAYA

    KUALA LUMPUR

    2012

  • ii

    UNIVERSITI MALAYA

    ORIGINAL LITERARY WORK DECLARATION

    Name of Candidate: NOR FARIZAH BINTI ABDUL HAMID

    I.C/Passport No: 850415-04-5106 Registration/Matric No: SGR080023 Name of Degree: MASTER OF SCIENCE Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): EFFECT OF SPERM AND OOCYTE FACTORS ON INTRACYTOPLASMIC

    SPERM INJECTION (ICSI) PERFORMANCE IN MICE AND GOATS

    Field of Study: ANIMAL BIOTECHNOLOGY

    I do solemnly and sincerely declare that: (1) I am the sole author/writer of this Work;

    (2) This Work is original;

    (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or

    reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this

    Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the

    making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the

    University of Malaya (“UM”), who henceforth shall be owner of the copyright in

    this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first

    had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any

    copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM.

    Candidate’s Signature Date

    Subscribed and solemnly declared before,

    Witness’s Signature

    Name: Date

    Designation:

  • iii

    ABSTRACT

    Selected sperm and oocyte factors related were evaluated in ICSI experiments involving

    mouse and goat species in the present study. The mouse oocyte collection was

    conducted through oviduct retrieval, while the goat oocytes were retrieved via LOPU

    technique. The effectiveness of sperm capacitation chemical treatment (Heparin and

    Theophylline) for 1 hour and sperm movement (Rapid and Slow) were identified in both

    species. For oocyte factors, the effects of post-hCG injection durations (13-15 hours and

    16-18 hours) in mice were studied. LOPU cycles (OR1, OR2 and OR3) and IVM

    durations (18-21 hours and 22-25 hours) were investigated in goats.

    In Experiments 1, the effects of different sperm capacitation chemicals and

    sperm movement of mouse sperm were studied. Mouse ICSI-embryonic development at

    early stages (2- and 4-cell stage) were improved by using Heparin compared to

    Theophylline (64.67±1.69% versus 60.50±1.21% and 50.55±2.01% versus

    45.09±1.60%, respectively). Both sperm movement factors had same potential to

    produce mouse ICSI-derived embryos.

    In Experiments 2, the effect of post-hCG injection duration in mouse oocytes on

    embryonic development rates were studied. Two different durations were significantly

    different (P

  • iv

    development (P>0.05). Rapid sperm movement gave better goat ICSI-embryonic

    development (4-, 8- cell and morula stage), whereby the respective values were

    56.62±4.69% versus 41.80±4.49%, 39.51±4.70% versus 24.24±4.05% and 9.21±2.85%

    versus 2.24±0.94%.

    Experiment 4 investigated the effect of LOPU cycles on the yield of different

    oocyte grades (Grades A, B, C, D and E) from 16 donors goat. Lower number of Grades

    D and E (31 and 19, respectively) oocytes were obtained compared to Grades A, B and

    C. Higher number of Grade C oocytes were obtained compared to Grades A and B from

    LOPU technique (153 versus 106 and 91, respectively). OR1 gave better quantity and

    quality oocytes, followed by OR2 and OR3 (number of oocytes per ovary were

    5.47±0.67, 3.94±0.44 and 3.09±0.50, respectively). In another sub-experiment,

    maturation rate of goat oocytes at 18 to 21 hours was higher than 22 to 25 hours of IVM

    duration (97.34±7.86% versus 89.01±2.41%, respectively). However, both IVM

    durations had the same potential in ICSI-derived embryo development.

    In conclusion, for mouse study, embryonic development can be achieved from

    Heparin-sperm capacitation treatment, using any sperm movement for 13 to 15 hours of

    post-hCG suntikan oocytes. For goat study, LOPU is good procedure to provide

    consistent high quality oocytes (Grades A, B and C) and it usage can be repeated on the

    same donor up to 3 times. Rapid sperm movement is a better choice for ICSI-embryonic

    development. Both sperm capacitation chemical treatments have the same potential to

    produce ICSI-embryonic development.

  • v

    ABSTRAK

    Faktor sperma dan oosit tertentu berkaitan telah dinilai dalam eksperimen ICSI

    melibatkan spesies mencit dan kambing dalam kajian kini. Pengumpulan oosit mencit

    dilakukan melalui perolehan oviduk, manakala oosit kambing diperolehi melalui teknik

    LOPU. Keberkesanan perlakuan kimia kapasitasi sperma (Heparin dan Theophylline)

    selama 1 jam dan pergerakan sperma (laju dan perlahan) dikenalpasti pada kedua-dua

    spesies. Bagi faktor oosit, kesan tempoh suntikan pasca-hCG (13-15 jam dan 16-18

    jam) pada mencit dikaji. Kitaran LOPU (OR1, OR2 and OR3) dan tempoh IVM (18-21

    jam dan 22-25 jam) disiasat dalam kambing.

    Dalam Eksperimen 1, kesan perbezaan kimia kapasitasi sperma dan pergerakan

    sperma dikaji. Perkembangan embrio-ICSI mencit pada peringkat awal (2- dan 4-

    peringkat sel) boleh diperbaiki dengan penggunaan Heparin berbanding Theophylline

    (64.67±1.69% berbanding 60.50±1.21% dan 50.55±2.01% berbanding 45.09±1.60%,

    masing-masing). Kedua-dua faktor pergerakan sperma menunjukkan potensi yang sama

    untuk menghasilkan embrio melalui ICSI.

    Dalam Eksperimen 2, kesan tempoh suntikan pasca-hCG terhadap oosit mencit

    terhadap kadar perkembangan embrio dikaji. Terdapat perbezaan ketara pada dua

    tempoh yang berbeza (P

  • vi

    kapasitasi sperma memberi potensi sama pada perkembangan embrio-ICSI (P>0.05).

    Pergerakan sperma yang laju memberi perkembangan embrio-ICSI yang lebih baik

    (peringkat 4-, 8- dan morula )berbanding pergerakan sperma yang perlahan, di mana

    nilai masing-masing ialah 56.62±4.69% berbanding 41.80±4.49%, 39.51±4.70%

    berbanding 24.24±4.05% and 9.21±2.85% berbanding 2.24±0.94%.

    Eksperimen 4, menyiasat kesan kitaran LOPU terhadap hasil gred oosit (Gred A,

    B, C, D dan E) yang berbeza daripada 16 kambing penderma. Oosit Gred D dan E

    adalah yang terendah (31 dan 19, masing-masing) diperolehi berbanding dengan Gred

    A, B dan C. Gred C menunjukkan hasilan oosit yang tertinggi berbanding oosit Gred A,

    dan B daripada teknik LOPU (153 berbanding 106 dan 91, masing-masing). OR1

    memberikan kuantiti dan kualiti oosit yang baik, diikuti oleh OR2 dan OR3 (bilangan

    oosit per ovari adalah 5.47±0.67, 3.94±0.44 dan 3.09±0.50, masing-masing). Pada sub-

    eksperimen yang lain, kadar kematangan oosit kambing pada tempoh 18 ke 21 jam

    adalah tinggi berbanding pada tempoh IVM 22 ke 25 jam (97.34±7.86% berbanding

    89.01±2.41%, masing-masing). Walau bagaimanapun, kedua-dua tempoh IVM

    mempunyai kadar potensi yang sama dalam perkembangan embrio perolehan ICSI.

    Kesimpulannya, bagi kajian mencit, perkembangan embrio boleh dicapai

    daripada perlakuan kapasitasi Heparin-kapasitasi sperma, dengan menggunakan mana-

    mana pergerakan sperma dengan mengguna 13 ke 15 jam tempoh suntikan pasca-hCG.

    Bagi kajian kambing, LOPU adalah prosedur yang baik untuk membekalkan oosit

    berkualiti tinggi secara konsisten (Gred A, B dan C) dan penggunaanya boleh diulang

    pada penderma yang sama sehingga 3 kali. Pergerakan sperm yang laju adalah pilihan

    yang lebih baik untuk perkembangan embrio-ICSI. Kedua-dua perlakuan kimia

    kapasitasi sperma memberi potensi yang sama untuk menghasilkan perkembangan

    embrio-ICSI.

  • vii

    ACKNOWLEDGEMENTS

    First of all, I would like to express my thanks and respect to my supervisor, Professor

    Dr Wan Khadijah Embong and co-supervisor Professor Dr. Ramli Abdullah for giving

    me chance, unfailing advice and motivation and continual guidance from the beginning

    of research until the completion of this dissertation. A lot of scientific advice,

    enthusias

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