small embryos? Ryan Waznik and Randall English Key Reproductive - - PowerPoint PPT Presentation

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

➢ Progesterone- hormone that is responsible for maintenance of pregnancy ➢ Corpus Luteum (CL) ○

• varian 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 F2𝞫 ○ lyses the CL and restarts estrus cycle ➢ Superovulation ○ production of multiple oocytes at one time with the use of follicle stimulating hormone (FSH)

Key Reproductive Terms

➢ 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

What is Embryo Transfer?

➢ 3 Steps of Embryo Transfer ○ Donor cow selection and superovulation ○ Flushing (collection) of embryos ○ Recipient female synchronization and transfer

Steps of Embryo Transfer

➢ Main Question: Can elevated progesterone levels rescue small blastocysts by increasing conceptus size?

What Questions are asked?

➢ 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.

➢ 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

Embryo Production

➢ 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.

Experimental Design (Figure 1)

➢ 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

Data Collection

➢ 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 6- 13, but only significantly on day 8.

Progesterone Levels (Figure 2)

➢ 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 Recovery (Table 1)

➢ 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

Conceptus Dimensions (Figure 3)

➢ No significant differences in luteal tissue weight ➢ PRID had a lower luteal area than all

• ther groups

○ is this surprising?

Measurements of CL (Figure 4)

➢ 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

Discussion

➢ 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

Limitations

➢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

Main Conclusions

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

Sources