ABT 103- ANIMAL BIOTECH.pdf

1. REPRODUCTIVE CYCLES OF FEMALE ANIMALS ESTRUS - n; Refers to the period of sexual receptivity of female mammals (synonymous to “heat”) ESTROUS - adj; Refers to the cycle of follicular development in female mammals (different from menstrual cycle) ANESTRUS - Happens when a female is not having r...

1. REPRODUCTIVE CYCLES OF FEMALE ANIMALS ESTRUS - n; Refers to the period of sexual receptivity of female mammals (synonymous to “heat”) ESTROUS - adj; Refers to the cycle of follicular development in female mammals (different from menstrual cycle) ANESTRUS - Happens when a female is not having repeated estrous cycles TYPES OF CYCLICITY POLYESTRUS - More than one estrous cycle per year ○ Ex. Queen (induced ovulatory), Cow, Sow SEASONAL POLYESTRUS - Cycles more than once in specific periods of the year ○ Ex. short day breeders (SDB) : sheep, goat deer long day breeders (LDB): horses, hamsters AVERAGE REPRODUCTIVE CYCLES SPECIES LENGTH OF LENGTH OF OVULATION LENGTH OF CYCLE ESTRUS PREGNANCY COW 21 days 18 hrs 11 hrs after 282 days polyestrus estrus EWE 17 days 29 hrs Near end of 148 days seasonal estrus SOW 21 days 48-72 hrs 35-45 hrs after 114 days polyestrus start of estrus MARE 21 days 4-8 days 3rd-6th day of 335 days seasonal estrus BITCH 6 months 9 days 4-24 days after 63 days diestrus start of estrus QUEEN 17 days induced 9 days induced 63 days ovulator CYCLE TYPES SPECIES TYPE OF FOLLICULAR OVULATION & CL Function CYCLE DEVELOPMENT CL FORMATION COW, EWE, Long Spontaneous Spontaneous Spontaneous SOW, MARE RATS, MICE, Short (4 Spontaneous Spontaneous Induced HAMSTERS days ave.) (prolactin) RABBIT, CAT, Induced Spontaneous Induced (LH Spontaneous MINK, FERRET, surge) OTTER, ALPACA ESTROUS CYCLE The female estrous cycle is a repetitive sequence of physiological changes in the reproductive tract brought about by hormonal interplay. It starts at sexual maturity and is interrupted by anestrus phases or pregnancies Hormones and factors involved: ○ Progesterone ○ Prostaglandin F2α (PGF2α) ○ Estradiol / Estrogen ○ Follicle stimulating hormone (FSH) ○ Luteinizing hormone (LH) ESTROUS CYCLE: PROESTRUS Decrease in progesterone due to regression of old corpus luteum (CL) into corpus albicans (CA) via the action of PGF2α (luteolysis). Maturation of one or two follicles due to positive feedback of LH and estrogen ESTROUS CYCLE: ESTRUS Ovulation usually occurs during estrus. A drastic increase in LH levels (LH surge) triggers the mature follicle to rupture thus releasing the ovum. The rupture follicle will then develop into a new CL. Estrogen levels starts to decline during this phase. ESTROUS CYCLE: METESTRUS Start of the luteal phase and is a period of sexual inactivity. The corpus luteum starts to develop during this phase.  Progesterone starts to increase as the CL continues to mature, thus preparing the uterus for implantation. ESTROUS CYCLE: DIESTRUS Period of optimum activity of the CL with increased levels of progesterone. If implantation of fertilized ovum occurs, progesterone will continue to be produced to support the gestation. If there is no implantation, the uterus will secrete PGF2α to stop progesterone secretion and force the system to undergo another cycle of estrus (back to proestrus). ESTROUS CYCLE: INDUCED OVULATION Induced ovulators like cats, rabbits, ferrets and camels require a stimulus (usually of physical copulation) in order to signal the anterior pituitary gland to create an “LH surge” which then triggers ovulation. In the case of the bitch, multiple matings create a greater and longer LH surge than single mating, thus increasing the chance of conception. ANESTRUS (factors: Pregnancy, Lactation, Pathology, Stress, Nutrition, Season) GESTATIONAL ANESTRUS ○ Progesterone during pregnancy has negative feedback effect on GnRH. Allows time for uterine involution. SEASONAL ANESTRUS ○ Due to effect of different photoperiods on the activity of the pineal gland. LACTATIONAL ANESTRUS ○ Suckling has been determined to lower blood LH levels, thus inhibiting ovulation. Prolactin inhibits GnRH release. NUTRITIONAL, PATHOLOGICAL & STRESS-RELATED ANESTRUS ○ Amount of body energy reserves and other existing physiological conditions greatly affect the hormonal balance of the animal which may disrupt the estrous cycle. For the estrous cycle, animals are only receptive to mating when they are in heat. Knowing at what point of the estrous cycle an animal is in will be very useful particularly for those who practice Artificial Insemination because they can determine the period that would allow for the highest chance of conception. The estrous cycle and menstrual cycle differ in two aspects: For estrous cyclers, the endometrial lining of the uterus is reabsorbed by the body if no embryonic implantation occurs. For menstrual cyclers, the endometrial lining is shed off if there is no implantation thus leading to the characteristic “menses”.  Unlike estrous cyclers, animals that undergo menstrual cycles do not exhibit distinct periods of sexual receptivity. Animals who have menstrual cycles (like humans) can be sexually active at any point of their cycles. 2. ARTIFICIAL INSEMINATION 1322 - Story of an Arab chieftain who allegedly stole stallion sperm from his adversary to inseminate his prized mare. 1776 - A technique for artificially inseminating dogs was developed by the Italian priest, Lazzaro Spallanzani 1799 - First recorded successful AI procedure in humans performed by British surgeon John Hunter. 1900 - Ilya Ivanov developed most of the techniques used today in AI. He started with breeding horses but controversially attempted to make human-ape hybrids. 1960s - Dairy cooperatives merge and form large companies that dominate cattle AI industry ARTIFICIAL INSEMINATION IN THE PHILIPPINE CONTEXT The dairy industry in the Philippines is still very small therefore AI services for dairy cattle is minimal. AI services for pigs and water buffalo are more prevalent around the country. Price range for water buffalo A.I. services (Php 300 – 1000) and is usually managed by select regional offices of the Department of Agriculture. Research in using AI is not just being conducted for animal production improvement but also for genetic resources conservation (e.g. native animals). ARTIFICIAL INSEMINATION: ADVANTAGES Genetic Improvement ○ Widespread use and availability of genetically superior sires ○ 1 bull can breed 500,000 cows in a lifetime ○ After death, semen can be used (Oldest frozen semen 40 - 45 years old) Rapid proof of sire ○ Progeny testing examines offspring for desired traits ○ With natural mating would only have 100’s of offspring Availability of sires ○ Sires anywhere in world Danger of bull (male) removed Disease reduction Crossbreeding ○ Can try without buying sire ○ Designer animals  Economics ○ Cost of sire genetics reduced ○ Sire maintenance cost reduced ARTIFICIAL INSEMINATION: DISADVANTAGES Estrus detection must be good. Trained inseminator is required. Bull semen the best, other species are more challenging to use. Use of poor male may increase if not tested well. Technology to store cooled or frozen semen is difficult to maintain. WHEN TO INSEMINATE VIA A.I. Detection of estrus ○ AI must be done as close to ovulation as possible. Time of insemination ○ Cattle (2X daily heat detection) 12 hours after observed in standing heat (AM - PM rule) Inseminate on the day of estrus Ovsynch, Co-Synch, Cidr - timed AI ○ Swine (2X daily heat detection) Sow - 24 and 36 hours after first seen in estrus Gilt - 12 and 24 hours after first seen in estrus ○ Sheep 12 to 18 hours after first seen in estrus ○ Horses Every second day beginning on day 3 of heat Breed when reach 40 - 45 mm follicle Breed 24 hours after human chorionic gonadotropin (hCG) injection hCG given when a >35 mm follicle is present Ovulation is 36 to 40 hours after hCG ○ Dogs Bitches ovulate around day 10 after they enter proestrus (discharge) or about 1 - 2 days of estrus. Ovulation can be detected by: LH assay (peak LH value + one day) Progesterone assay (>5 ng/ml) Cytology of vaginal smear (>50% cornified cells) Remember oocytes in the canine are ovulated as 1° oocytes and must mature in the oviduct to a 2° oocyte before fertilization.  Fresh or cooled semen, inseminate 2 days after ovulation detected and again 48 - 72 hr latter. Frozen semen, inseminate on day 5 - 7 after ovulation Uterine insemination better than cervical A.I. TECHNIQUES: CATTLE Rectal-Vaginal Approach A.I. TECHNIQUES: HORSE Vaginal Approach A.I. TECHNIQUES: PIG USE OF SPECIALIZED CATHETER A.I. TECHNIQUES: DOG USE OF SPECIALIZED CATHETER FACTORS AFFECTING CONCEPTION RATE Time of insemination ○ If after ovulation then get aging of oocytes Exception is the dog # of sperm inseminated Fertility of males Skill of inseminator 3. CRYOPRESERVATION CRYOPRESERVATION Process of preserving organelles, cells, tissues, and even whole organisms by keeping them in very low temperatures to effectively “halt” all metabolic processes and prevent organic deterioration. Requires an understanding of the interaction of biological components (particularly fluids and membranes) and low temperatures in order to prevent “cryoinjury” or damage due to freezing. CRYOPRESERVATION an integral part of methods to control animal reproduction. Long-term storage of cells. Genetic conservations and Cryo-banking of animal species. Adjunct to the treatment of animal diseases in the future-Cryonics. Partial solution to the problem of ageing (space exploration). Bob Nelson - (President of the Cryonics Society exploration). of California) demonstrating the cryonic process in 1967 Cryobiology of cells The major hurdle for cells to overcome at low temperatures is the water-to-ice phase transition. ○ Cell injury at fast cooling rates is attributed to intracellular ice formation. ○ Slow cooling causes osmotic changes because of exposure to highly concentrated intra- and extracellular solutions or to mechanical interactions between cells and the extracellular ice COMPONENTS OF CRYOPROTECTIVE COCKTAIL - The goal of a cryoprotective cocktail is to support osmotic efflux of water and increase intracellular viscosity to minimize post-thaw degradation of the cells. Cryoprotectant (CPA) ○ compounds that lower down the freezing point of cells Sugars ○ large, hyper-osmotic, permeating or non-permeating compounds Macromolecules ○ high molecular weight compounds that make the solution viscous METHODS OF CRYOPRESERVATION Equilibrium freezing: ○ Slow or conventional freezing technique Non-equilibrium freezing technique ○ Rapid or Quick-freezing technique ○ Ultra-rapid freezing technique Vitrification technique ○ Formation of a stable, glass-like state under ultra-low temperatures WHAT ARE THE CHALLENGES IN CRYOPRESERVATION? Intracellular and extracellular ice crystal formation ○ Formation of ice crystals may lead to damage to the cellular membrane or it may cause cell lysis due to rapid expansion of water molecules as it freezes. Cryoprotectant toxicity before and after freezing ○ CPAs such as glycerol, dimethyl sulfoxide or propanediol are toxic at high concentrations. Osmotic stress ○ Maintaining minimal osmotic shock on cells during freezing and thawing is important to preserve the viability of cells. Effective cryopreservation must protect the cell against the known deleterious effects of cooling and warming An understanding of the different components/solutions and their roles in cryopreservation will enhance the success of cryopreservation procedures 4. MULTIPLE OVULATION & EMBRYO TRANSFER STEPS IN FERTILIZATION Attraction (hormonal chemotaxis) and capacitation. Binding to corona radiata. Tunneling through the Zona pellucida via acrosome reaction. Cortical reaction. Continuation of ovum meiosis II. Fusion of pronucleus Mitosis (zygotic cellular division) MOET Performed to increase the reproductive rate of high performing females. Reduces the generation interval of superior dams. Involves: ○ Oocyte isolation and maturation ○ Sperm preparation ○ Sperm capacitation ○ Fertilization ○ Embryo Development HORMONES USED IN MOET DONOR ○ FSH – stimulates follicle production ○ PMSG – stimulate follicle production ○ GnRH – preovulatory surge of LH, ovulation RECIPIENT (estrus synchronization) ○ Progesterone – Inhibits ovulation ○ Prostaglandin – Induces destruction of CL METHODS IN SUPEROVULATION Intramuscular injection of pregnant mare’s serum gonadotrophin (PMSG or eCG) + PSGF2α or an analogue of it, or; 8-10 intramuscular or subcutaneous injections of FSH at 12-hour intervals + PSGF2α or an analogue of it *PMSG results to larger ovary, generally double the volume of one treated with FSH due to its very long half-life (five days) in cattle (that of FSH is several hours) resulting in continued recruitment of follicles after ovulation, very high progesterone levels and, probably, abnormalities in ovum transport. The basic instrument for non-surgical recovery is the Foley catheter Generally 18- to 24-gauge sizes are used. It is best to use as large a catheter as can be introduced easily to achieve good rates of flow. Most people prefer two- way catheters, one passage for air and one for fluid, because rates of flow are higher than with three-way catheters, which have two smaller passageways for fluid. STAGE OF EMBRYO AT RECOVERY Compacted morula ○ DAY 5-7 Cavitating morula ○ DAY 7-8 Expanded blastocyst ○ DAY 7-9 EMBRYO SEXING Hy Antigen ○ Associated with male cells PCR and Detection of Y and X DNA LIMITING FACTORS IN MOET Costly hormones Labor intensive protocols and expertise Poor superovulatory response Poor pregnancy response Large Calf Syndrome