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Drm science lecture MENOPAUSE AND CRYOPRESERVATION

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MENOPAUSE
CRYOPRESERVATION
OESTROUS CYCLE

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Neuroprotective and CNS effects- insomnia, fattigue
Tibolone-synthetic norsteroid derivative-estrogenic,
progestrogenic, a...

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Dysfunctional Uretine Bleeding -Mainly progestins
Dysmenorrhoea- mainly NSAIDS, cyclic estrogens
Acne and hirsuitism-cycli...

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Drm science lecture MENOPAUSE AND CRYOPRESERVATION

  1. 1. MENOPAUSE CRYOPRESERVATION OESTROUS CYCLE
  2. 2. Neuroprotective and CNS effects- insomnia, fattigue Tibolone-synthetic norsteroid derivative-estrogenic, progestrogenic, and weak androgenic properties Dose-2.5mg OD ERT in primary ovarian failure- Estrogens in cyclical pattern- Ethinyl estradiol-0.01mg- 3 wk for 4 m -0.02mg- 3wk for 1yr
  3. 3. Dysfunctional Uretine Bleeding -Mainly progestins Dysmenorrhoea- mainly NSAIDS, cyclic estrogens Acne and hirsuitism-cyclic estrogens ERT in primary ovarian failure- Estrogens in cyclical pattern- Ethinyl estradiol-0.01mg- 3 wk for 4 m -0.02mg- 3wk for 1yr Estrogen adverse effects Gynacomastia, Feminisation, Prostatic Ca, Migraine, Breast tenderness, Amenorrhea, Gall stones, hepatic dysfunction C/I- diabetes, hepatic failure, thromboembolic disorder
  4. 4. 1. Oral contraceptives 2. HRT to limit estrogen’s effects on the endometrium 3. Uterine Bleeding disorders -DUB 4. Premature labor (decrease uterine contractions) 5. Stimulate Appetite in AIDS or cancer patients 6. Endometriosis-danazol(non-progestrogenic, non- estrogenic, but exhibits hypoestrogenic, hyperandrogenic, cause atropy of endometrium) 7. Premenstrual syndrome 8. Threatened abortion 9. Endometrial Carcinoma
  5. 5. Progressive accomplishment of alterations in genital tract characteristic of estrus, metestrus, diestrus and proestrus produced by changes in ovarian hormones Occurs in non-primate females Estrous period or estrus Period of sexual receptivity in female Commonly called "heat".
  6. 6. Animals Length of cycle Length of heat Sheep 16-17 days 24-36 hours goat 21 days 32-40 hours pig 19-21 days 48-72 hours cow 20-22 days 18-24 hours horse 19-25 days 4-7 days
  7. 7. Proestrus (FOLLICULAR PHASE) Days 17 to 21 of estrous cycle of cow Ovarian follicles are growing rapidly Increase in Estrogen secretion by growing follicles Decrease in Progesterone from corpus luteum (CL) Mucosal layers of vagina and uterus multiply Estrus Day 1 of estrous cycle of cow Period of sexual receptivity Standing Heat Surges of FSH and LH from anterior pituitary gland pig, sheep and horse ovulate during this phase
  8. 8. Metestrus Days 2 to 4 of estrous cycle of cow Short transitional phase after ovulation with decreasing Estrogen and increasing Progesterone concentrations Ruptured follicle is reorganizing into a Corpus Leuteum(CL)Cow ovulates during this phase Diestrus (LUTEAL PHASE) Days 5 to 16 of estrous cycle of cow  Continuation of luteal phase in species with long estrous cycles CL becomes fully developed and Progesterone exerts a dominant influence on oviducts, uterus, vagina, pituitary and hypothalamus
  9. 9. 1. 3. 2. ENDOCRINE REGULATION OF ESTROUS CYCLE
  10. 10. I. Introduction - The term menopause is derived from Greek Meno (months) and pause (cessation). The word means cessation of menstruation. - Cliamacteric which is by dictionary definition is period of life when fertility and sexual activity decline. It is a wide term leading to: Pre Menopause Peri Menopause Post Menopause
  11. 11. - It is 3-5 years period before menopause with increase frequent irregular anovulatory bleeding followed by episodes of ammenorrhea and intermittent menopausal symptoms. Menopause: - The point in time at which menstrual cycles permanently cease. It is a retrospective diagnosis after 12 months of ammenorrhea women classified as being menopause. - Mean age – 51 years.
  12. 12. The number of primordial follicle decline even before birth but dramatic just before menopause. Increase FSH, LH from about 10 years before menopause. Close to menopause: There will be -anovulation -inadequate Leuteal phase → decrease progesterone but not astrogen level → lead to DUB and endometrial Hyperplasia - at menopause dramatic decrease of Estrogen→ menstruation ceases and symptoms of menopause started. But still ovarian stroma produce →small androstenedione and testosterone but, main postmenopausal estrogen is estrone produced by Peripheral fat from adrenal androgen.
  13. 13. Hot flushes cutaneous vasodilation - occurs in 75% of women - more severe after surgical menopause - continue for 1 year - 25% continue more than 5 years Urinary Symptoms - urgency - frequency - nocturia Psychological changes decreased level of central neurotransmitters - Depression, Irritability, Anxiety - Insomia - lose of concentration
  14. 14. Cryo is Greek word. (krayos – frost) It literally means preservation in “frozen state.” The principle - to bring plant cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant. It can be done : Over solid carbon dioxide (at -79 degree) Low temperature deep freezer (at -80 degree ) In vapor phase nitrogen (at -150 degree) In liquid nitrogen (at -196 degree) Cryopreservation
  15. 15. Once the material is sucessfully conserved to particular temperature it can be preserved indefinately. Once in storage no chance of new contamination of fungus or bacteria. Minimal space required. Minimal labour required. Cryopreservation
  16. 16. The cryopreservation technique followed by the regeneration. It involves following steps : Selection of material. Addition of cryoprotectant. Freezing. Storage in liquid nitrogen. Thawing. Washing and reculturing. Measurement of viability. Regeneration Mechanism of cryopreservation
  17. 17. Genotypic drift Senescence leading to extinction of cell line Transformation to tumor related properties Contamination Distribution to others Saving reagents, time Equipment failure such as incubator Cross-contamination by other cell lines
  18. 18. Sex determined in pre-implantation embryos Approach- either invasive or non invasive Splitting of sexed embryos First successful embryo sexing done by Gardner, 1968 in rabbits by cytological method (Barr body observation)
  19. 19. Non invasive Methods-- Immunological assay of HY antigen Quantification of X-linked enzyme Differential growth of male & female embryo Invasive Methods-- Cytogenetic analysis observing Barr bodies chromosome analysis Y-specific DNA probe Y-specific DNA primer & PCR
  20. 20. Barr body forms after certain stage in embryo depending on species Performed at blastocyst stage Affected normal embryonic development in some case outdated now Not applicable in domestic species Gardner, 1968
  21. 21. Sexing from Trophoblast biopsy, day 12 -- 15 Accuracy 58.5--68% sexed embryo at 6-7 days but low efficiency & low survival rate reported Slides prepared at metaphase stage Depends on how many cells at metaphase stage Takes an expert 5 hours to process 12 - 15 embryo
  22. 22. H-Y detection used for murine, bovine, porcine embryo. Detection as early as 8 cell stage. Can have two approaches cytotoxic or immunofluorscent. In pigs detected only after removal of zona pellucida. Accuracy 84% in cattle, 85% in goat, 81% in pigs. Immunofluorescent Assay detection stage specific Monoclonal and polyclonal antibodies has equal effect Poor quality embryo show fluorescence unrelated to presence of antigen
  23. 23. Revolutionized the technique of embryo sexing Reduced time requirement Increased efficiency Embryos have been successfully sexed in a number of farm animals by using this technique
  24. 24. Embryo sexing done for cattle, sheep, pigs, horses, goats, buffalo & humped cattle Differential growth rate not applicable to choose embryos Single cell is sufficient for sexing Rapid sexing within 2 hours by using multiplex PCR they used BOV-97M & bovine 1.715 satellite DNA sequence Non Electrophoretic method for PCR sexing reduced time requirement to less than 2 hours
  25. 25. Altering the male & female sex ratio in farm animals Increase in milk & meat production Getting quality bulls Conservation of rare breeds of farm animals

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