Effect of bovine blastocyst size at
embryo transfer on day 7 on
conceptus length on day 14: Can
supplementary progesterone rescue
small embryos?
Ryan Waznik and Randall English
Key Reproductive Terms
➢ Progesterone- hormone that is responsible for maintenance of pregnancy
➢ Corpus Luteum (CL)
○ ovarian structure that secretes progesterone after ovulation
➢ Human Chorionic Gonadotropin (hCG)○ induces ovulation of a dominant follicle that forms an accessory corpus
luteum (CL), thus increases circulating P4 concentration slowly
➢ Progesterone releasing intravaginal device (PRID)
○ exogenous progesterone insert that
○ increases P4 rapidly
➢ Prostaglandin F2𝞪
○ lyses the CL and restarts estrus cycle
➢ Superovulation
○ production of multiple oocytes at one time with the use of follicle
stimulating hormone (FSH)
What is Embryo Transfer?
➢ 10,000-12,000 heifer calves are produced per year due to embryo transfer
(ET)
➢ First calf produced in 1951
➢ A surgical or nonsurgical technique used to transfer embryos from a donor
cow to a recipient cow
○ used to produce superior female genetics
○ multiple offspring from donor
Steps of Embryo Transfer
➢ 3 Steps of Embryo Transfer
○ Donor cow selection and
superovulation
○ Flushing (collection) of
embryos
○ Recipient female
synchronization and
transfer
What Questions are asked?
➢ Main Question: Can elevated progesterone levels
rescue small blastocysts by increasing conceptus size?
➢ How do the treated cow’s
progesterone levels compare to
the controls for the first two
weeks after estrous?
➢ Is there a relationship between
the size of an embryo at
embryo transfer and the size of
the conceptus it forms after a
week of growth?
Main Hypothesis
The present study was designed to test the hypothesis
that supplemental P4, through hCG administration on
Day 2 or insertion of a PRID between Days 3 and 5,
would promote elongation of a small blastocyst with a
low total cell number at the time of transfer, that is,
P4 “priming” of the uterine environment can overcome
the decreased developmental potential of embryos with
small cell number.
➢ Sub-hypothesis:
○ By increasing conceptus size on Day 14, we can
increase pregnancy rates.
Embryo Production
➢ Immature Cumulus oocyte complexes (COCs) were aspirated from
slaughtered heifers and cows
➢ Matured for 24 hours in vitro
➢ Mature COCs were inseminated in vitro from the frozen-thawed sperm of
a single bull
➢ 20 hours after insemination, the embryos were isolated from cumulus
cells and accessory sperm, and were cultured until day 7
➢ Classified by size at day 7 as large or small based on estimated cell
number from diameter lengths
➢ Embryo transfer into recipient heifers
Experimental Design (Figure 1)
➢ P4 (days -8 to -2) and PG (day -3) were used to synchronize estrous cycles
to standing at day zero in recipient heifers.
➢ Treatment groups received either hCG at day 2 or PRID from days 3 to 5.
➢ At day 7, blastocysts transferred to the recipient heifers via ET.
➢ The heifers were slaughtered at day 14 for conceptus recovery.
➢ Blood was sampled from days 0 to 14.
Data Collection
➢ Blood samples collected daily from day 0 to day 14
○ measured progesterone concentrations
➢ Day 7 blastocysts collected
○ diameter measured
○ some stained with bisbenzimide to measure cell number and
➢ Conceptus recovery on day 14
○ recovery rates of conceptuses versus number of embryos transferred
were measured
○ length, width, and weight were measured
➢ CL dissection of slaughtered heifers
○ weighed and measured for dimensions
Progesterone Levels (Figure 2)
➢ Control small and control
large had similar
progesterone levels
➢ PRID had Significantly higher
progesterone on days 4-5
➢ hCG group yielded higher
progesterone from days 613, but only significantly on
day 8.
Conceptus Recovery (Table 1)
➢ Each heifer received
10 embryos
➢ PRID had significantly
less % recovered from
transfers than control
large
➢ hCG had significantly
less % recovered from
yielding heifers than
all other groups
Conceptus Dimensions (Figure 3)
➢
➢
➢
➢
Measured in mm for length and width, and mm^2 for area
PRID had significantly greater elongation and area
Small control had smaller conceptus dimensions than large (not sig.)
hCG had greater length and area than small control
Measurements of CL (Figure 4)
➢ No significant differences in luteal tissue
weight
➢ PRID had a lower luteal area than all
other groups
○ is this surprising?
Discussion
➢ Increased elongation in day 14 conceptuses was seen in treatment,
especially PRID
➢ The treatments did not show an increase in recovery of conceptuses
○ Though quantity did not increase, quality via measurement of
elongation could lead to fewer miscarriages later on and higher birth
rates in treatments
➢ PRID method shortens cycles, but can help conceptus elongation
➢ Combined methods of PRID and hCG have shown to help cycles, but have
not been evaluated for effect on conceptus elongation
➢ Further studies could look more into the utilization of combined
techniques to see its potential usefulness in elongation without as much
damage done to the CL
Limitations
➢ High variation in embryo size
➢ Short term study
○ Why was the study short term?
■ In order to evaluate short term elongation
○ Cannot answer whether bigger is better
➢ Only measured 1 hormone level
○ What other compound concentrations might be relevant to the
study?
■ INFT, mentioned to be affected by progesterone
■ Estradiol and inhibin A, products of the CL
Main Conclusions
➢ Supplementation of P4 can rescue small
embryos
➢ Blastocyst cell count at day 7 is correlated
to day 14 conceptus length
➢ The PRID method has shown to have a
marked increase in day 14 conceptus length
Sources
http://upload.wikimedia.org/wikipedia/commons/6/6b/E
mbryo,_8_cells.jpg
http://www.bairnsley.com/Web%20Photos/AI%20%20ET%20flush%20diagram.jpg
http://www.davis-rairdan.com/embryo-transfer.htm
http://msucares.com/pubs/publications/p2681.pdf
http://www.ncbi.nlm.nih.gov/pubmed/12604631
http://www.jbc.org/content/269/40/24826.full.pdf