Chpater 18 Altering Reproductive Processes

    * Improved animal reproduction: From higher reproductvie rates, more genetic gain, and better reproductive management

    A. By seeking better management of natural reproductive processes

    B. By enhancing genetic and reproductive mangement by means of altering reproductive processes including:

      a. Artificial insemination
      b. Synchronization of estrus
      c. Superovulation
      d. Embryo transfer
      e. Promoting twinning
      f. In vitro sperm capacitation and oocyte maturation
      g. In vitro fertilization
      h. Sex control
      i. Embryo cloning
      j. Transgenic animals
      k. Induced parturition
      l. Castration
      m. Vasectomized males and androgenized heifers

    18-1 Synchronization of estrus

    A. Definition: To have a number of females in estrus during a very short period of time

    B. Advantages:

      a. To schedule livestock handling and breeding to fit into a work schedule
      b. To reduce time-consuming job of detecting estrus
      c. To shorten breeding season
      d. To group animals into desired parturition patterns so that intensive care may be provided for limited periods

    18-1-1 Progestin method

    A. Physiological bases: Preventing release of GnRH, FSH and LH by negative feedback action of exogenous progesterone for sufficiently long time ≧ to allow corpora lutea to regress in order to obtain estrus synchronization following exogeneous progestin withdrawal simultaneously in a number of females.

    B. Methods of progesterone administration:

      a. Progestins: Progesterone and synthetic progestins as: Flurogestone acetate(FGA) and Melengesterol acetate (MGA) etc

      b. Adminstration methods: 1) Oral administration, 2) Ear implant as Norgestomet: See FIGURE 18-1, and progesterone-releasing intravaginal device (PRID: a silastic coated coil)

      c. Duration of administrtion: 16 days in cow and 14 days in ewes

    C. Estrus syncronization: 80-90% females: Estrus occurred over 4-day period from 2 to 6 days following progestin withrawal

    D. CR% in synchronized females: 15% lower than in spontaneously estrous females

    E. Timed-insemination in synchronized females: See TABLE 18-1

    Some treated cows ovulate without overt signs of estrus and conceive to

    timed-insemintions. But CR% is lower than in cows bred during detected estrus

    18-1-2 Prostaglandin method for cows

    A. Physiological bases:

      a. Prostaglandin (PG) F2メ administered as a single injection during days 5 through 17 of estrous cycle causes luteolysis and subsequent return to estrus in 36 to 72 hrs.
      b. No response from Day 1 through Day 4:

        1) CL may not have sufficient PGF2メ receptor sites to respond to normal levels of PGF2メ.
        2) Metabolic pathways may not have developed at this point to allow complete response: because some cows respond to a very large dose of PGF2メ.

      c. No effective after Day 17: CL is usually regressing spontaneously
      d. Response at an unaccepatable rate from Day 5 to Day 7: See TABLE 18-2

    B. Methods of estrus synchronization by PGF2メ

      a. Dose: a single i.m. injection of 25 mg PGF2メ or 0.5 mg cloprostenol (an PGF2メ analog): High percentage is deactivated, mainly in lung, prior to reaching CL

      b. Effectiveness:

        1) Serum progesterone level: fall rapidly within 24 hrs.
        2) Serum estrogen level: rise within 24 hrs.
        3) Preovulatory LH peak: occur within 3 days
        4) Estrus: occur at about time of LH peak
        5) Ovulation: occur about 24 hrs. after onset of estrus

      c. Practical 4 alternative methods:

        1) Inject cows only with CL of 5 days or more in age by rectal palpation, time-inseminate estrous cows 80 hrs. following treatment, and retreat remaining cows 12 days later, and then time-inseminate estrous ones

        2) Treat all cows, inseminate estrous cows, and retreat all remaining cows 12 days following the first treatment, and then time-inseminate estrous cows

        3) Check estrus for 5 days, inseminate estrous cows, treat remaining         animals on day 5, and inseminate at the ensuing estrus: 65 to 70% CR

        4) Treat all cows. wait 12 days, retreat all cows, and then either time-inseminate at 80 hrs or inseminate estrous cows only (Remember that only cycling cows will respond to PGF        2メ treatment)

        * Physiological base of time-insemination 80 hrs after treatment: Many females begin to show estrus 60 to 72 hrs after treatment, and the optimum period for insemination is 8 to 18 hrs after onset of estrus: So higher CR% can be expected when inseminating at detected estrus.

      d. Results of estrus synchronization experiments: See TABLE 18-3
      e. CR% improvement by GnRH treatment at Days 12 to 14 following AI with PG-induced estrus: See Lajili et al (1991) Theriogenol. 36:335-347

    18-1-3 Combination of progestins and prostaglandins for cows

    Progstins adminstered for 7 days + PGF2メ on day 6: From an experiment in Holstein heifers: 82% and 100% of treated animals showed estrus within 17- and 32 hr-period, respectively.

    * A practical measure for shortening postpartum open days in cows:

    1) Observe estrus intensively from 45 days postpartum
    2) Treat PGF    2メ to cows which do not exhibit estrus until 50 days postpatum
    3) Retreat PGF2メ 12 days later to cows which did not respond
    4) Treat cows with GnRH at 12 to 14 days post AI
    5) Confirm cows of negative early pregnancy disgnosis (EPD) at 22 days post AI
    6) Treat PGF    2メ to negative EPD cows at 32 days post AI

    18-1-4 Synchronization of estrus in mares

    PGF2メ injection from days 6 post-ovulation to day 18 of the cycle: Return to estrus in 4 to 5 days and ovulate 10 to 12 days post-injection

    18-1-5 Synchronization of estrus in sows

    A. PGF2メ is not practical because sow's CL will not respond until day 12 of the cycle

    B. Altrenogest, a synthetic progestin, when fed for 14 or 18 days, will bring 90 to 95% of a group sows into estrus within 6 days after withdrawal, with equal subsequent CR%

    18-1-6 Synchronization of estrus in ewes

    Estrus synchronization by using progestins or PGF2メ: Detrimental effect to sperm and sperm transport will occur in the cervix

    18-1-7 Synchronization of estrus in does

    A. PGF2メ is available but more research is needed.

    B. FGA for 17 to 22 days with PMSG treatment on removal of progestin: Good synchrony and CR%

    18-2 Superovulation and embryo transfer(ET)

    A. Cow, ewe and doe: An average of 12 ovulations: expected by superovulation; In cows: 7.9 embryos collected nonsurgically, 60 to 70% of superovulated ova: normal embryos (4 to 5 normal embryos/ superovulation)

    B. Mare: No reliable superovulation

    C. Sow: Can be accomplished, but 12 to 20 spontaneous ovulations

    D. Calves: Variable responses and very low fertilization rates

    18-2-1 Techniques of superovulation

    A. Ewes: A single s.c. injection of 600 - 1,000 IU PMSG on day 12 or 13

    B. Cows:

      a. Twice-daily injections of 5 mg FSH for 4 or 5 days starting days 8 - 13 of the cycle
      b. Twice-daily injections of a diminishing dose of 5,4,3 and 2 mg FSH for 4 or 5 days starting days 8 - 13

      * Estrus will be induced on days 5 of superovulation regime with a single i.m. injection of 25 mg PGF2メ on the 3rd day ( at the same time FSH injection number 6 is given).
      * Multiple AI should be performed over a 2 day period.
      * Repeat superovulation in cows: 10 repeat superovulations(￥ 3 ovulations) possible in the interval of 60 days, but most become refractory much sooner due to an immune reaction by foreign FSH or eCG protein

    C. Sows: A single i.c. injection of 750 - 1,000 IU eCG on day 15 of the cycle and a single i.m. injection of 500 IU hCG at the onset of estrus

    18-2-2 Bovine embryo collection and evaluation

    A. Embryo collection

    * Embryo collection date: Schedule a day 7 collection (estrus = day 0), Identify and palpate all recipients that showed estrus 6, 7, or 8 days earlier

      a. Surgical collection: See FIGURE 18-3

        1) Lapaotomy to expose reproductive tract
        2) Inject fluid into uterine horn and force it through oviduct and collect embryos at infundibulum
        * High embryo recovery rate, but surgical trauma and resulting adhesions

      b. Nonsurgical collection:

        1) Use of Foley catheter: See FIGURE 18-4:


        Figure 18-4 Foley catheter used to flush embryos from the uterus by nonsurgical means. 

        2- way or 3-way catheters and catheters of different diameter: See Ref. Figure 25 of Curtis, 1989: CATTLE EMBRYO TRANSFER PROCEDURE

        2) Insert catheter into right or left uterine horn using stylet and cervical dilator: See Ref. Figures 26 and 28 of Curtis,1989.

        3) Inflate catheter's balloon by air injection through "c" and hemostat at "d" of Ref. Figure 27 of Curtis, 1989.

        4) After removal of stylet, flush uterine horn with PBS as TABLE 18-4 using Y-junction tubing and Em-Con filter of Ref. Figures 23 and 24 of Curtis, 1989

    B. Embryo evaluation

      * Accurate embryo evaluation: important for successful ET
      * Some characteristics of normal embryos:

      a. Compactness of the cells: compact rather than loose mass of cells
      b. Regularity of shape: spherical shape rather than oval,etc
      c. Variation in cell size: blastomeres of similar size
      d. Color and texture of cytoplasm:neither very light nor dark color
      e. Presence of vesicles: some moderate sized vesicles, rather than granular or unevenly distributed
      f. Presence of extruded cells: without any dissociated cells
      g. Normal embryo size
      h. Regularity of zona pellucida: with empty perivitelline space and of regular diameter and even (neither wrinkled nor collapsed) zona pellucida
      i. Proper stage of development for age: See FIGURE 8-1

    18-2-3 Embryo storage and transfer

    A. Embryo storage for shorter than 10 hours

      a. Embryos just collected: Maintained at near body temperature in flusing media from collection to transfer to recipients
      b. For storage for 2 to 10 hours:

        1) Media should contain 20% heat-treated serum.
        2) Embryos should be transferred to fresh, sterile medium every 2 hours to help control bacterial contaminants.
        3) If 37” cannot be maintained constantly, it may be better to allow embryos to cool to room temperature.

    B. Embryo cryopreservation: Read further: Niemann(1991). Theriogenol. 35:109-124

      a. Slow freezing using a programable embryo freezer: (Procedure used in Niemann's lab.)

        1) One-step addition of a 1.4 M glycerol solution with 20 min equilibration at room temperature.

        2) Loading into 0.25 ml straws: The freezing solution is sucked into the straw, separated into 3 segments by small air bubbles, the mid-segment containing the embryo.

        3) Transfer to the ethanol bath precooled to -7 ”　

        4) Induction of crystallization (= seeding) with a pair of supercooled forceps followed by a holding period for 5 min.

        5) Slow cooling by embryo freezer from -7 ”to -28 ” at o.3 ”/ min.

        6) Slow cooling from -28 ” to -35” at 0.1”/ min.

        7) Plunge into liquid nitrogen

        8) Thawing in air at room temperature

        9) Removal of glycerol using sucrose in 2 steps.

        * CR% following nonsurgical transfer of frozen embryos: > 50%

      b. Vitrification: Rapid freezing without the use of an expensive freezing machine

        1) Definition: Physical process by which a highly concentrated solution of cryoprotectants solidifies during cooling without formation of ice crystals rapid freezing without the use of freezing machine

        2) Results with livestock embryos are still limited, but bovine, ovine and caprine morulae and blastocysts have been vitrified successfully and following transfer given birth of normal offspring.

    B. Embryo transfer

    * Embryos must be transferred to the uterine horn ipsilateral to ovary containing CL of recipients having their estrus cycles synchronized with the donor

      a. Surgical transfer: Bovine embryos of blastocyst stage will be transferred into the uterine horn, exposed by laparotmy, with syringe fitted with a 21-gauge needle: not practical

      b. Nonsurgical transfer:

        1) By puncturing uterine horn with a long hypodermic needle through vagina: not practical
        2) By carefully passing 0.25 ml straw inseminating-gun into uterine horn through cervix: Somewhat lower CR%, compared with surgical transfer, but used in most commercial embryo transfer

    18-2-4 Promoting twinning

    A. Methods for twinning

      a. Superovulation

        1) Space limitations from several embryos result in embryo mortality to reduce the number of fetuses to 1,2 or 3.

        2) Too many implantation frequently occur and later result in abortions

      b. Transfer of 2 IVF embryos: See Jiang et al(1991). Theriogenol. 35: 216: CR%: > 60% and twinning rate: >50% from nonsurgical transfer to 56 recipient heifers

    B. Disadvantages for twinning

      a. Short gestations accompanied by small and weak calves
      b. Freemartins from heterosexual twins
      c. More stress on the cows- dairy cows produce 10% less milk in the lactation following twin birth
      d. Increased reproductive problems: High incidence of retained placenta, cystic ovairies, etc
      e. Insufficient milk for 2 calves in most beef cows: Retarded calf growth