FERTILISASI : PERTEMUAN ♀ x ♂ (Prose's penyatuan 2 gamete) Untuk mencapai inti ovum spermatozoa hrs melewati 1. sel granulosa 2. Zona pellusida 3. dinding sel ovum (membran vitellin) • Kepala sperm menempel pd zone pellucida (receptor zp) • Ab antizona: memblok penempelan sperm • Ikatan ZP dpt dihambat dg Ab antisperm atau glikoprotein yg diekstrak dr ZP. • Glycosyl transferase, proteinase dan glycosidase pd lapisan membran plasma kepala sperm dpt menghasilkan ikatan pada ZP3 PENETRASI SPERM Untuk masuk ke dalam sel ovum, spermatozoa terlebih dahulu harus menembus: (a). Massa kumulus (bila masih ada) (b). Zona pelusida (c). Membran vitellin Pertautan kepala sperma dengan permukaan membran vitellus. aktivasi sel ovum Diepididymis (10 – 15 hr) Maturasi tergt dari sekresi epididymis dan waktu transport Motility/hyperactivation Di Ampula, Sp aktif bergerak (ampula mengandung zat spt: bicarbonate, pyruvate, asam amino bebas,oxygen, karbohidrate, steroid, Co2 dan nucleosides) produk sel-sel mukosa ampula Bag kepala sp mengandung enzim hyaluronidase yg berfungsi mencairkan asam hyhaloronate diantara sel-sel granulosa Sp menembus dd zp dan kepala sp bersentuhan dg membran vitellin dan terjadi reaksi zona (untuk mencegah sp lain masuk) Reaksi zona zat yg dilepaskan oleh granula kortika yg terdapat diluar membran vitellin RZ : jelas pada domba, tapi tdk jelas pada babi (antara m.vitellin dan zp sering ditemukan lebih dari 1 ekor sp.) dan kelinci ± 200 sp Pd hw yg tdk mempunyai RZ, Vitellin Blok mempunyai RZ dan VB sangat penting karena ovum dan sp adl sel-sel haploid Haploid + haploid Diploid (berkemb normal) Supplementary sperm (sperma suplemen): Pada ruang ZP lebih dr 1 sp • Mengetahui lifespans (fertilitas) ovum dan sperm • Waktu ovulasi (tgt sp.hewanya) • Kosent sperm ( hanya 1.000 – 10.000 sp mencapai istmus) dan hanya 10 - 100 sp yg mencapai ampula setelah 4 sampai 12 jam Seleksi pada uterotubal junction, isthmus (babi) vagina, servik, uterus (ruminansia Mencegah polispermi Setelah Fertilisasi (Reaksi Acrosoma) Intracellular calcium oscillations Cortical reaction Zona reaction Block of polyspermy Istilah: supernumenary sperm (Sperma Supernumerik) : bila sudah terjadi RZ dan VB, tapi dlm vitellus didapat lebih dari 1 sp Polispermia fertilisasi gagal fertilisasi (jmlh kromosum > dr normal) Triploid kebunt. mati PENJIWAAN SPERM DAN OVUM • Kepala sp menyentuh membran vitelin • Reaksi membran ditandai, adanya tonjolan kecil pd membran • Kepala dan seluruh tubuh sp masuk kedlm sitoplasma sel ovum • Membran plasma yg menjadi pembungkus sp lebur menjadi satu dg membran vitellin • Pengkerutan protein dan pembelahan inti sel ovum yg terakhir • Hasil pengkerutan: dikeluarkannya cairan kedalam rongga antara zp dan vitellin • Hasil pembelahan inti sel ovum menghasilkan polar bodi Kepala sperma terlepas dg bagian lainnya dan menggembung Period of Cleavage • zygote • blastomeres • morula • Fertlisasi Zygote (gamet) 2 cell, 4 cell, 8 cell, 16 cell dan 32 cell (Dlm Zona Pelluucida) 32 cell (stadium morulla) – Cairan terlihat terkumpul diantara cel morulla Balstocoele – Tubuh embrio terlihat terbagi 2, karena ada bag sel yg tumbuh membentuk lapisan tipis dibag permukaan – Bag luar disebut Trophoblast Plasenta – Bag dalam inner cell mass Mahluk baru 32 cell (stadium morulla) • Cairan terlihat terkumpul diantara cel morulla Balstocoele • Tubuh embrio terlihat terbagi 2, karena ada bag sel yg tumbuh membentuk lapisan tipis dibag permukaan • Bag luar disebut Trophoblast Plasenta • Bag dalam inner cell mass Mahluk baru • Morolla masuk uterus Blastocyst yg mempunyai trophoblast • Fungsi trophoblast : menyerap cairan yg mengandung nutrisi bagi embrio • Pada beberapa mamalia zp pecah setelah Blastocyst menyentuh endometrium Implantasi • Cairan uterus memp. peran sangat penting dalam menunjang kehidupan embrio menjelang implantasi (spt; Glysin, alanine, taurine dan glutamine) Time of Events in Early Embryonic Development spesies Gamet Longevity (Hours) • Sperm • Ovum Cattle Horse Sheep Swine 30-40 20-24 72-120 6-8 30-48 16-24 34-72 5-10 Ebryonic Development (days) • 2- cell • 4-cell • 8-cell • Blastocyst • Hatching 1 1,5 3 7-8 9-11 Blastocyst Transport to Uterus Hours Cell stage 72-84 8-16 Blastocyst Elongation (days) 13-21 1 1,5 3 6 8 140-144 Blastocyst - 1 1,3 1,5 6-7 7-8 66-72 8-16 11-16 0,6-0,8 1 2,5 5-6 6 46-48 4 11-15 Initial Placentation (days) Birt (days) 22 17 278-290 335-345 15 145-155 13 112-115 AWAL PERKEMBANGAN ENBRIO IMPLANTASI Proses implantasi adl proses yg berlangs scr bertahap 1. 2. 3. 4. Persentuhan embrio dengan endometrium Terlepasnya Zona pellusida Pergeseran atau pembagian tempat (polytocous) Pertautan trophoblast dengan epitel endometrium Pecahnya Zp: - ada sebelum menyentuh endometrium (beberapa mammalia) - setelah menyentuh endometrium (marmot) akibat adanya juluran-juluran protein yg berasal dr trophpbalst (akibat reaksi dr embrio) Hewan Polytocous: mekanisme penyebaran embrio disebut dingan mekanisme kontraksi uterus Pecahnya Zp: • ada sebelum menyentuh endomet (bbrp mammalia) • setelah menyentuh endometrium (marmot), akibat adanya juluran protein yg berasal dr trophpbalst (akibat reaksi dr embrio) Hewan Polytocous: mekanisme penyebaran embrio disebut mekanisme kontraksi uterus (Spacing embrio) Implantasi terjadi: bila embrio telah bertautan dengan endometrium dan tempatnya tdk berubah SIFAT IMPLANTASI • Embrionya terbenam dalam kripta endomet (seluruh trophoblast berhub. sangat erat dng dinding kelenjar endomet) hewan pengerat (rodentia) • Emb. hanya bersentuhan dng epithel endomet (pertautan terjadi karena penjuluran protein trophoblast ke endomet sehingga seluruh tubuh embrio masih berada dlm lumen uterus sifat implantasi mengambang (sulit ditentuka Domba: implantasi ± hr ke 10-22 setelah IB Sapi: hr ke 11 – 40 Model implantasi kedua sp ini adl sangat rawan Implantasi pd babi • • • • • Setelah 1,5 – 2 hr di tuba : embrio masuk utereus Spacing embrio (pengaturan tempat embrio) 10-15 hr Terlepasnya zp (sebelum implantasi) Stl Implantasi: trophpblast tumbuh cepat Nutrisi untuk embrio berasal dr uterus Histotrophe (susu uterus) • Terjadi hubungan yg erat antara trophoblast dengan endometrium terbentuk plasenta Domba • 3hr post ovulasi morulla dan memasuki lumen uterus • Morulla blastula • Blastula melayang-layang dlm histotrophe (17 hr post ovulasi) Masih dapat di flushing • Db. Mempunyai karunkula dan tersebar diseluruh • • • • • permukaan endomet Jumlah karunkula terbanyak adl pada curvatura mayor dr cornua uteri dan mempunyai demensi lebih besar drpd di bagian apex cornua uteri Karunkula berbtk mangkok (cotyledonaria) jml ± 90 buah Terjadi persentuhan antara trophoblast dg epitel karunkula Sel-sel trophoblast masuk dalm cell vili pada karunkula pertautan yg sangat erat Hr ke 30 implantasi selesai Sapi • Hampir sama dg db (karunkula) • Jumlah embrio hampir sama 1-2 buah Bedanya: faktor waktu dan kemungkinan terjadinya anastomosa bila kembar (tidak pd domba) • Anastomosa (kembar ♂♀) Freemartin (majir) • Implantasi lebih lambat yi. ± 33 hr • Karunkula berbtk spt bunga kol (cotyledonaria) jml 100 -110 KOTILEDON / CHORION Endometrium Endometrium KARUNKULA Membran Slip Plasentoma PADA KUDA • Blastocyst dapat hidup dlm susu uterus sampai 60 hr • Ukuran embrio tdk mengalami perkemb yg cepat (± 5 cm) • > 60 hr, trophoblas tumbuh vili-vili • Umur 14 mgg (3,5 bln) terjadi pertautan implantasi terjadi PLACENTATION Tenunan tubuh embrio dan induknya, yg terjalin waktu pertumb. embrio untuk keperluan penyaluran makanan dr induk ke anak dan zat buangan dr anak ke induk. Kebuntingan muda: jr tubuh embrio paling luar menjadi Amnion, allantois, chorion dan kantong kuning telur (yolk sac) Amnion: bag yg menyelubungi fetus dibagian paling dalam Khorion: bag yg menyelubungi fetus dibagian paling luar Allantois: bag yg terdapat diantara amnion dan khorion • Lapis sel allantois dibag dalam menjadi satu atau berhimpitan dg sel-sel membrana amnion • Lapis luar berfusi atau berhimpitan dng sel-sel membrana khorion • Arteri dan vena dr plasenta ke tubuh embrio berada pd lapisan membrana allantois dan khorion • Adanya ruang amnion yg berisi cairan yg konsistensinya agak kental Fungsi cairan amnion: mengurangi getaran atau goncangan dr luar tubuh induk dan sbg tempat penampungan zat buangan dr embrio mll urethra. • Cairan allantois konsistensinya lebih encer fetus mengikuti perubahan posisi induk memungkinkan bagi Fungsi : sebagai tempat pembuangan urin melalui urachus (sal urin yg menghubungkan kantong urin dan kantong allantois melalui tali pusar. • Kantong kuning telur tehenti tummbuh setelah kantong amniom dan allantois terbentuk seutuhnya Placentation Reptilian Chorioallantoic Placenta Chorioallantoic placenta of the lizard Mabuya Mammalian Placentation • Transitory placentae exist in most species – Yolk sac – Chorionic – Chorioamniotic • Chorioallantoic – formed by chorion, • Chorioallantoamniotic – final placenta Chorionic Connective Tissue • Embryo travels down tube and enters uterus • Embryo interacts with wall of uterus – Implantation – Maternal recognition of pregnanc Trophoblast • expands and forms placental tissues • 2 types •Cytotrophoblast •Syncytiotrophoblast • Cyto - main region of the placenta • Syncytio- invasive tissue Uterine Response •Decidualization •Inflammation response endometrium overgrowsembryo Inflammation response • endometrium overgrows embryo • decidualization Placental Shapes • Diffuse – Horses, camels, pigs, dolphins • Zonary – Carnivores (raccoon, dog, cat) • Cotyledonary (placentomes) – Cows, sheep • Discoid – Primates, rodents, rabbits, insectivores Discoid Cotyledonary Zonary Diffuse • Zonary – Dog, Cat Chorion Paraplacenta (pigmented) STRUKTUR HISTOLIOGIS FETUS INDUK Kapiler Endothel Endothel Jar. Ikat Epitel Chorion Epitel Endometrium Jar Ikat STRUKTUR HISTOLIOGIS • Macamnya dibedakan berdasarkan jumlah lapisan tenunan sel yg memisahkan aliran darah induk dan anak • Ada 5 struktur scr histologis 1. epiheliochoreale 2. syndesmochoreale 3. endotheliochoreale 4. hemochreale 5. hemoendothelia Ephiteliochoreale: tdp pd tipe P difusa (kd) darah induk dan anak dipisahkan oleh 2 lapis epitel, 2 lapis endotel, dan 2 lapis tenunan pengikat yg masin-masing berasal dr endo dan trophoblast syndesmochoreale: tdp pd tipe P. cotyledonaria (sp,kb,db) darah anak dan induk dipisahkan oleh 3 lap. Tenunan sel yi. Endotel, tenunan pengikat dan tenunan epitel trophoblast (dr anak). Dari induk hanya endothel endotheliochoreale : tdp pd tipe P. zonaria (aj, kc) Hemochreale : tdp pd tpe plasenta diskoidales (man, mencit, tikus, marmot dan kelinci) mempunyai lapisan pemisah darah anak dan induk lebih tipis dan dipisahkan oleh 3 tenunan lap sel yg berasal dr trophoblast (endotel, t.pengikat dan ep. trophoblast) Hemoendothelia : (hemo = darah, endothel = epithel p.darah) darah induk dan anak hanya dipisahkan oleh 1 lapis tenunan sel yi endothel dr p.darah anak (tdp pd kelinci dg tipe P.diskoidale) Placental Classification • Based on number of layers separating fetal and maternal blood Syndesmochorial (Epitheliolchorial) (Ruminants) Binucleate giant cells (unique) • Transfer placental lactogen • Secrete pregnancy specific protein B • Steroidogenesis Endotheliochorial (bitch, queen) Complete erosion of endometrial epithelium and interstitium; maternal capillaries exposed to chorionic epithelium Hemochorial (primates, rodents) Maternal blood in contact with chorionic epithelium Hemoendothelial (rabbit, rat, guinea pig) Maternal blood in contact with chorionic capillaries Placental Transfer • Simple diffusion – Water, gases • Facilitated diffusion – Glucose, amino acids • Active transport – Na, K, Ca • Not transferred: – Immunoglobulins (species dependent) – Proteins – Lipids • Transferred – Steroid hormones – Toxic substances (pharmaceuticals, heavy metals) – Microorganisms (viruses, bacteria) Placental Hormone Production Maintenance of pregnancy Stimulation of ovarian function Stimulation of mammary gland Promotion of fetal growth and maturity Assist in parturition Placental Hormones: Chorionic Gonadotropins • Equine (eCG) – LH like activity in horses – FSH activity in others – Produced by endometrial cups Placental Hormones: Chorionic Gonadotropins • eCG – LH-like – Secondary CL’s – Accessory CL’s Placental Hormones: Chorionic Gonadotropins • Human (hCG) –LH like activity – Present in urine of pregnant women –Used to induce ovulation, luteinization Human Hemochorial Maternal Recognition of Pregnancy • Prevention of luteolysis – Anti-luteolytic – Luteotrophic • Maintenance of elevated progesterone Cow, Ewe Trophoblastic protein - Interferon tau Cow: bIFN-τ (btp-1) Ewe: oIFN-τ (otp-1) - Produced by trophoblast, d 13-21 post ovulation Inhibits oxytocin receptor synthesis by endometrium Also promotes protein synthesis by endometrial glands for embryo survival Sow • Estradiol – Produced d 11-12 post ovulation – Increases prolactin receptors – Promotes exocrine secretion of PGF – Stimulates uterine contractions to distribute fetuses – Minimum 2 conceptuses per horn required Mare • Embryonic mobility • Embryo doesn’t • elongate Day 12-14 post ovulation “Implantation” Initial attachment (d) Attachment complete (d) Cow 25 40 Ewe 16 30 Sow 12 18-20 Mare 45-50 100-120 Placenta • Metabolic exchange between dam and conceptus – Chorion (villi) – Endometrium • Transient endocrine organ – Maintenance of pregnancy – Preparation for parturition, lactation – Initiation of parturition BENTUK MAKROSKOPIS PLASENTA BENTUK MAKROSKOPIK PLASENTA • Khorion adl bagian lap terluar dr trophoblast • Trophoblast tumbuh menjalar menyelimuti seluruh • • • • • • • permukaan endo Terbentuknya jar plasenta karena penjalaran trophoblat (tgt dr spesies) Sapi dan domba: lap luar trophoblast bersentuhan langs dg endo. Kuda dan babi: plasenta terbentuk dr hub. Superfisial dr sel epitel endo dan epitel khorion Tipe plasenta pd sp,db dan kb: Cotyledonaria Tipe pada babi dan kuda: difusa Tipe pd anjing: zonaria Tipe pd kera: diskoidalis Periode dr mulai fertilisasi sampai kelahiran normal 3 periode kebuntingan 1. Periode ovum : mulai fertilisasi sampai implantasi) 2. Periode embrio : implantasi sampai pembentukan 3. alat2 tubuh bag. dalam) Periode fetus : periode terakhir dari terbentuknya alat tubuh bag dalam, extremitas sampai lahir Lama kebuntingan: masing-masing sp berbeda Perbedaan individu dalam spesies disebabkan oleh: . Faktor genetik . Faktor sosial dan lingkungan SIRKULASI PLASENTA • 2 sirkulasi yg paralel yaitu sirkulasi fetus dan maternal • • • • • (induk) Pada plasenta epithelichoreale, darah ini tidak tercampur Darah yg mensuplai plasenta adalah darah arteri dan vena uterina Arteri umbilicus membawa darah dr fetus ke plasenta Darah kembali ke vena umbilicus dr plasenta ke fetus Aliran darah fetus dan maternal bisa countercurrent, concurrent dan crosscurrent (aliran yg berbeda) SIRKULASI DARAH FETUS • Pada prinsipnya sama dg hewan dewasa, kecuali oxygenation terjadi pada plasenta bukan pada paru-paru • Sirkulasi fetus juga diperkirakan mempunyai aliran darah oksigen langsung kejaringan 1. Ductus venosus liver ke darah terbesar vena umbilicalis dr dalam Parturition Initiation of Parturition • • • • Fetal maturity Stage 1: Initiation of myometrial contractions Stage 2: Expulsion of fetus Stage 3: Expulsion of fetal membranes • Fetal pituitary-adrenal axis – Fetal stress – Release of fetal ACTH – Stimulates fetal adrenals to produce corticoids Fetal corticoids Promotes synthesis of: •17a hydroxylase •17-20 desmolase •Aromatase Increased E2 Decreased P4 Removal of ‘P4 block’ Increased secretions of reproductive tract Hormonal Changes that Control Parturition Hypothalamus Steps Fetal ACTH causes - Fetal anterior pituitary gland Corticotropic Releasing Hormone CR Time of parturition is F controlled by maturation of the hypothalamuspituitary-adrenal axis Adrenalcorticotropic Fetal Corticosteroids Hormone (ACTH) causes Removal Blocks Parturition Progesterone levels Infusion Stimulates placental production induces oxytocin receptor or CL regression) parturition Fetal in myometrium Adrenal Production of Estrogens Estrogen by placenta Corticosteroid (Increase) PGF2a production by uterus - Pine-needle abortion in cattle - cause by a corticosteroid-like product Glucocorticoid Redrawn from Liggins, G.C. 1969. In Foetal Autonomy Progesterone (Decrease) Prostaglandin F2a (Increase) Significance of initial hormonal changes Progesterone - removes block on uterine contractions. Allows myometrial muscle fibers to work together in bundles Estrogen - makes uterus more responsive to induction of contractions i.e., more irritable and smooth muscle tissue stimulation. Induction of oxytocin receptors Events just Prior to Parturition: 1. Pelvic ligaments soften - Tail head sinks due to estrogens and relaxin. 2. Cervix softens and begins secreting stringy mucus - estrogens and relaxin. Increased water content in cervical tissue and cervical plug is removed 3. Swelling of vulva. Collagenase breaks down collagen which also widens the pelvis 4. Udder swells - fills with first milk and due to edema: Prolactin, Estrogens and glucocorticoids 5. Fetus moves into proper position - resting on thorax, front feet and head facing the cervix Stages of Parturition: I. Dilation of Cervix. (2-6 hours, cow) Heifers 12 hours Uterine contractions become coordinated and regular Synchronized Estrogen & PGF2a induced This occurs because progesterone has declined Fetus pushed against cervix - amnion dilates cervix Stimulates oxytocin release Chorioallantoic membrane may break 1st Water Bag Pressure of fetus in cervix stimulates oxytocin release and reflex Ferguson contractions of abdominal muscles. Reflex Contractions force the calf legs and head to spread cervix II. Expulsion of Fetus (.5-2 hours, cow) Longer than 2 hours considered to have dystocia Strong uterine contractions due to synergistic actions of high estrogen, PGF2a and oxytocin Strong abdominal muscle contractions Amnion ruptures - mucin lubricates vagina - vestibule 2nd Water Bag Fetus passes through vagina - vestibule. Cause of death in 6.4% of calf losses on average Three Barriers in Delivery of a Calf 1. Head 2. Shoulders 3. Hips Sow Delivery of Piglets Length of Stage II in sow is 2 to 4 hours Delivery of piglets is usually between 8 to 45 minutes between each one. Delivery will alternate piglets between horns Last 1/4 of horn in large litters increases the chance of stillborn Length of umbilical cord Uterine horn contracts to shorten distance after delivery of each piglet III. Expulsion of the Placenta (4-5 hours, cow) Uterine contractions continue Blood forced from cotyledon villi - shrinkage separates cotyledon and caruncle Prolapsed uterus cow tired need to give Contractions push placenta out oxytocin Causes of retained Placentas: -Infections caused adhesions between cotyledon and caruncle -Calving stress - twins or calving difficulty - edema of cotyledon - won't separate from caruncle -Weak uterine contractions - villi won't shrink Oxytocin Milk fever - Calcium low Parturition: Stage I • Increased myometrial activity • Fetal positioning • Cervical relaxation/dilation • Oxytocin release • Rupture of chorioallantois Parturition: Stage II • Estrogen stimulates secretory activity • Fetal activity (response to hypoxia) stimulates myometrial activity • Cervical stretching (pressure) causes oxytocin release • Eventually fetus is delivered Parturition: Stage III • Release of fetal membranes Care of retained placentas: Do not manually remove from uterus; uterine damage greater than infection of retained placenta Do not pull placenta out even in Mare! Check to see placenta is fully intact in Mare or you need a Veterinarian to clean her out. Don’t breed on foal heat if there is a problem Infuse Bovine uterus with tetracycline and systemic injections of penicillin until placenta passes ~ 2-4 days. Don’t give mare tetracycline as this will cause inflammation!! Can treat cow with either PGF2a or oxytocin (ergonovine) to expel the infection Hormonal changes cause: 1. Final maturation of fetus Cortisol - stimulates lung surfactant 2. Expansion of birth canal Initiates parturition and lung development 3. Maternal behavior 4. Synthesis and ejection of milk 5. Initiation of uterine contractions 6. Termination of pregnancy Fescue toxicity problems: Ergot causes inhibition of prolactin release and thus milk production Prolactin completes final mammary development and milk secretion Fetal Corticoids • Stimulate PGF synthesis – Stimulates myometrial activity – Causes luteolysis Puerperium • ‘Reproductive repair’ – Uterine involution • Myometrial contractions • Expulsion of lochia • Endometrial repair – Resumption of ovarian activity – Elimination of bacterial contamination Myometrial Contractions • Stimulated by oxytocin • Removal of debris and fluid from uterus • Compress uterine vasculature, minimize hemorrhage • Reduce size of uterus Lochia • Normal discharge • Duration varies with species Lactation • Mammary Gland – Isometric growth until puberty – Allometric growth post-puberty • Hormonal influence Mammary Gland Development • Repeated estrous cycles result in development of mammary glands • Alveoli – Secretory tissue – Develop under progesterone • Ducts – Estrogen – Prolactin – Somatotropin Mammary Gland Development • Last trimester – Lobules develop in alveoli – Milk secretion dependent on • Prolactin • Adrenal cortical hormones • Placental lactogen Mammary Gland Secretions • Colostrum – Immunoglobulins – Growth factors – Temporary secretion • Milk – Composition differs among species Mammary Gland Involution • Decreased suckling – Pressure atrophy • Capable of rapid cessation • Critical for milk production after subsequent pregnancy Milk Ejection • Secretion vs. Ejection – Secretion into alveoli – If not secreted, pressure atrophy occurs • Ejection – Neuroendocrine reflex