Larry  Lanzon,  Lander  Veterinary  Clinic   August  17,  2015  

2015  ARSBC,  UC-­‐Davis   1  

Applied Reproductive Technologies in Practice You Can Do It!

Larry Lanzon D.V.M., M.P.V.M.

Overview �  Artificial insemination �  Embryo transfer �  Ultrasonography �  Ovum pickup and in vitro fertilization �  Stem cell oocyte production

Responsibilities �  Herd Health �  Expected Outcomes ◦  AI ◦  ET

2048 recoveries mean 11.5 ova/embryos, average 6.2 viable embryos (Looney, CR, 1986.) 4440 recoveries mean 15.3 ova/embryos, average 7.7 viable embryos (LVCET 2015, unpubl.)

Artificial Insemination

 

Animal Selection

•  Genetically and economically valuable females •  Seed stock producer •  Club calf producer •  Commercial producer •  Marketable progeny •  Decreased costs

Estrus Detection Detection in donor and recipients ◦  Donor breeding ◦  Recipient estrus synchrony with transferred embryo.

Detection Methods:

•  Cowboy on a horse •  Paint Stick® or spray paint •  Heat Patch® or Estrotect® patches

•  Automatic systems (commercially available)

Heat Watch®

Afimilk®

AccuBreed®

SensOor®

Larry  Lanzon,  Lander  Veterinary  Clinic   August  17,  2015  

2015  ARSBC,  UC-­‐Davis   2  

Synchronization of follicular waves

•  GnRH •  Prostaglandins •  CIDRs

Senger, P. L. (1997). Pathways to pregnancy and parturition.

Current Conceptions, Inc., 1615 NE Eastgate Blvd.

Insemination

•  Timing •  Semen handling

Animal Handling •  Good Facilities

•  Flow

•  Disposition Score •  Pen Score

Range of 1-5 •  Exit Score

Time/Distance

Carroll, J. Methods of assessing the stress level of cattle in order to bring about positives changes in reproduction. Society for Theriogenology. 2015. San Antonio, Texas.

Grandin, T., Review: Reducing handling stress improves both productivity and welfare. The professional Animal scientist, 1998. 14(1): p. 1-10.

Ultrasonography

Ultrasonography �  Donor reproductive exam �  Ovum pick up for in vitro fertilization �  Superovulation response �  Cow reproductive exam �  Bull reproductive exam

U/S - Cow exam Complete exam �  Uterus ◦  Pregnancy ◦  Fetal sex ◦  Estrus ◦  Abnormalities

�  Ovaries ◦  Follicle ◦  Corpus luteum ◦  Abnormalities

 

Larry  Lanzon,  Lander  Veterinary  Clinic   August  17,  2015  

2015  ARSBC,  UC-­‐Davis   3  

U/S - Bull exam Complete Examination �  External ◦  Spermatic cord ◦  Panpiniform plexus ◦  Epidydimus - head ◦  Epidydimus - body ◦  Epidydimus - tail ◦  Testis

�  Internal ◦  Prostate ◦  Ampullae ◦  Seminal vesicles

Embryo Transfer

ET - Application

•  Superior females with superior males •  Herd dynamics •  Repopulation •  Genetic reservoir •  Research •  Import and export

ET - Process �  Selection of donor and recipients �  Synchronization of donor and recipients �  Superovulation of the donor female �  Detection of estrus in donor and recipients �  Breeding of the donor �  Recovery of the embryos from the donor �  Transferring embryos to the recipients �  Freezing of the embryos

Selection Criteria Donor �  Progeny success in ET �  Calf >50 days old �  No periparturient disease �  BCS 2.5-3.5 �  Cycling �  High level of fertility �  Dry

Recipient �  Proven production �  Virgin heifers, or ≤ 7 years �  Calf >60 days of age �  No periparturient disease �  BCS >2.5 �  Cycling

Donor superovulation

�  Follicle Stimulating Hormone ◦  Reference estrus ◦  First follicular wave ◦  Second (day 9.5) and third follicular wave (day 10.5)

�  CIDR® ◦  GnRH à 2 day FSH start

Larry  Lanzon,  Lander  Veterinary  Clinic   August  17,  2015  

2015  ARSBC,  UC-­‐Davis   4  

Donor superovulation

•  Injection Timing •  FSH IM b.i.d. 10-12 hrs interval for 4 days

•  Prostaglandin with last 2 FSH injections

•  CIDR® removed at last prostaglandin injection

•  Insemination: •  2 doses 10-12 h standing •  2 doses 10-12 h later

ET - Response Variability �  Age �  Breed �  BCS �  Season �  Follicular wave size

Nutrition

Armstrong, D.T.,Theriogenology. 55(6): p. 1303-1322. Donaldson, L.E. Theriogenology, 1984. 21(6): p. 1013-1018.

Jaiswal, R., et al.,. Theriogenology, 2009. 72(1): p. 81-90. Mapletoft, R.J. IVIS Reviews in Veterinary Medicine,2006.

Singh, J., et al. Theriogenology, 2004. 62(1): p. 227-243.

ET - Embryo Recovery

•  Surgical Recovery •  Nonsurgical Recovery

•  Syringe method •  Gravity flow method

•  Solutions •  Commercially available •  Lactated Ringers with added surfactant

•  Recovery filter •  Microscopic dish search

ET - Embryo Processing

Embryo Stage •  Stage 1: Unfertilized •  Stage 2: 2-12 cells •  Stage 3: Early morula •  Stage 4: Morula •  Stage 5: Early blastocyst •  Stage 6: Blastocyst •  Stage 7: Expanded blastocyst •  Stage 8: Hatched blastocyst •  Stage 9: Expanding hatched blastocyst

Manual of the International Embryo Transfer Society, 4th Ed

Embryo Grading

•  Grade 1: Excellent or good •  Grade 2: Fair •  Grade 3: Poor •  Grade 4: Dead or degenerating

Larry  Lanzon,  Lander  Veterinary  Clinic   August  17,  2015  

2015  ARSBC,  UC-­‐Davis   5  

ET – Transfer of Embryos

•  Fresh embryo: •  Recipient preparation •  Embryo preparation •  Embryo Transfer

•  Previously frozen embryo: •  Recipient preparation •  Embryo preparation •  Ethylene glycol cryoprotectant •  Glycerol cryoprotectant

ET - Cryopreservation

•  Straw labeling •  Embryo preparation

•  Ethylene glycol •  Glycerol •  Vitrification

•  Embryo Freezing •  Frozen embryo storage

Photos: WL Walker

Other Technologies

Ovum Pickup (OPU) In Vitro Fertilization (IVF)

�  Donor selection �  Valuable donor not producing with ET �  Stimulated and non-stimulated �  Response variability �  Process �  Time and Temperature

OPU and IVF Results

 01/2014  -­‐  06/2015  Total   DAIRY   BEEF  

   Number  of  OPUs   92   41   51  

   Total  oocytes  recovered   937   546   391  

                                                                                         per  cow   10.2   13.3   7.7  

   Total  oocytes  matured   757   430   327  

                                                                                           per  cow   8.2   10.5   6.4  

   Total  G  1  &  2  oocytes  matured   512   290   222  

                                                                                           per  cow   5.6   7.1   4.4  

   Total  Embryos  produced   259   131   128  

                                                                                           per  cow   2.8   3.2   2.5  

%  Total   34%   30%   39%  %  Grade  1  and  2  Oocytes   51%   45%   58%  

Stem Cell Oocyte Production

�  The next ART?

�  Less invasive: animal welfare & reproduction

Purified oocyte germ cells capable of proliferating in vitro when transplanted into ovaries of recipient mice generate fully functional oocytes that fertilize to produce viable embryos and offspring.

Larry  Lanzon,  Lander  Veterinary  Clinic   August  17,  2015  

2015  ARSBC,  UC-­‐Davis   6  

Take Home �  Responsibilities �  Complete exam �  Design programs �  Embrace technologies

“tools for improved genetics and production”

Larry Lanzon DVM, MPVM Lander Veterinary Clinic Embryo Transfer 2930 Lander Ave. Turlock, CA 95380 209 678 3335 phone 209 634 0643 fax lvc_et@yahoo.com