Commercial freezing of bovine embryos in straws

THERIOGENOLOCY

COMRCIAL FREEZIffi OF BOVINE EMBRYOSIN STRAWS Joseph M. Wright Veterinary Services, P.O. Box 598 Castroville, Texas 78009

Medina Valley

P.C.

Abstract Bovine embryos were frozen comnsrcially in clear double length l/2 cc French straws with the wick and powder plug in the center of the straw. One-haIf of the double length straw serves as a handle and contains a color coded l/4 cc straw around which an adhesive backed label has been applied. After plunging into liquid nitrogen, straws are transferred into goblets on canes while under liquid nitrogen. The straws are stored in the liquid phase of a nitrogen tank and canes containing straws are not transferred from one container to another unless the goblet containing the straws is full of liquid nitrogen. Embryos held for longer than 4 hours after collection prior to freezing showed a steady decline in pregnancy rate related to the length of time held prior to freezing. The percentage of embryos thawed and then evaluated as being transferrable was related to the quality of the embryos prior to freeze (Grade l-93.6%, Grade 2-87.0%, Grade 3-63.8%). There was no statistical difference in pregnancy rates obtained from prefreeze Grade 1 embryos when conparing advanced blastocysts f45.2%), blastocysts f38.7%), early blastoclyst (43.1%) and advanced morula 141.6%).

INTRODUCTION Embryo freezing began at Medina Valley Veterinary Services in 1980 utilizing glass anpules and a Planer P.T.C. R202/200R freezer.a Problems occurred with labeling the anpules and poor heat seals that resulted in exploding anpules during thawing procedures. Only one pregnancy was obtained out of the first 50 en&ryos thawed. A decision was rrede to start over and solve the problems utilizing the French straw as the freezing container. With experience, the pregnancy rate has shown a steady increase from 10% in 1981 to 40% for en-&ryos frozen and thawed between July 1, 1983 and June 30, 1984. A total of 3252 embryos have been frozen since July of 1980 and 1922 were thawed by our staff prior to July 1, 1984. Four different freezing units have been utilized for the work. The purpose of this

paper is

to present

what we feel

is

the mst

practical way to freeze embryos in straws on a commercial basis. Considerations are given to methods of labeling, storage, records, inventory, shipment of frozen en&ryos and evaluation of freezing equipment.

aPlaner

Products Ltd., Middlesex, England

JANUARY

1985 VOL. 73 NO. I

17

THERIOGENOLOGY

The

French

Straw

as

a Freezing

Container

The French straw has proven it’s value in the packaging of frozen semen and has been well adapted to use in the non-surgical transfer of However, there are four important differences in the bovine embryos. freezing of embryos in straws as conpared to semen that nest be These factors are: considered. 1. Labeling 2. Air spaces inside the straw 3. Transferring straws to goblets for storage 4. Possible contamination by alcohol if an alcohol freezer is utilized. When

considering

length of the

l/2 cc straw.a

Figure

1

wick and (Figure

these

factors,

powder 1 I.

straw

l/2 MDIA

SEAL

MEDIA

CONTAINING

cc

USED

WICK

we with

COUBLE TO

Ai’&

EMQ YO

have the

LENGTH

SEAL

chosen wicks

COLORED STRAW

PLUG

k

use

a double

to

the

center

STRAW LABEL

POWDER

to moved

1/2cc INSIDE

SEAL HANDLE

+Jl

1.

Labelinq A comxrcial freezer is justified when freezing semen in straws because of the large nu&er of straws from each ejaculation. However, when freezing en&ryos, one can hand label the few straws required in much less time than required to set the type, print the straws, and Unfortunately labeling of straws by clean up the printing equipment. hand requires a fine point marking pen and a steady hand to include the information recomnended by the International Etiryo Transfer Society (IPTS! and required by breed associations. We started freezing etiryos in the l/4 cc straw which has In order to considerable less surface area than the l/2 cc straw. acconwwdate labeling and provide a handle to simplify transferring frozen embryos to goblets, we placed the etiryos in a l/4 cc straw and labeled a l/2 cc straw which was then heat sealed to the l/4 cc straw. this allowed us to color code the In addition to providing more space, different straws by utilizing different colored l/2 cc straws. This was fairly effective; however, the sealed junction was very brittle when placed in liquid nitrogen and would occasionally break off if not handled carefully. Because of this possibility, some labeling was required on both straws.

a Continental

IX

Plastic

Corp.,

Uelavan,

Wisconsin

JANUARY

1985 VOL. ‘3 NO. I

THERIOGENOLOGY

length 112 cc straw there was no brittle By going to a double junction to break or leak and allow nitrogen into the straw. A l/4 cc straw easily fits inside the handle portion of the double length straw and allows for color coding by utilizing different colored l/4 cc straws. Since it is easier to write on a flat surface than on the round surface of a straw, an adhesive backed label a is utilized. This 3 l/2 inch by 19/32 inch label when cut in half longitudinally fits around the l/4 cc straw inside the handle portion of the double length straw. This provides sufficient space for labeling requirements and has allowed us to put sire information on the label which is optional under JETS In addition, recomnendations, but required by some breed associations. this label is sealed inside the 112 cc straw, which protects it from loss and the solvent properties of alcohol. 2. Air spaces inside the straw When semen is frozen the entire straw is filled with the semen. However, when embryos are frozen, air spaces are included to prevent the embryos from sticking to the wick plug on one end or from being removed with the sealed end when it is cut off. The double length l/2 cc straw further complicates this because the handle end of the straw is filled with air. When

the sealed straw is cooled during the freezing process and the air spaces contract more than eventually stored in liquid nitrogen, liquid and cause a vacuum within the straw. If the seals are inperfect or weak they can leak and allow liquid nitrogen to enter the straw. Subsequently, when the straw is warmed during the thawing process, the liquid nitrogen rapidly expands and turns to gas causing internal This results in an exploding straw or the liquid pressure in the straw. containing

the embryos

being

forced

through

a crack

in a seal

with

loss

or damage to the embryo. This is a significant problem and probably accounts for rmny of the cracked zonas and damaged or lost embryos at thawing. In addition, if the frozen column of media containing the embryos moves before the ice crystals thaw, the ridged crystals can cause damage to the zona pellucida or the embryo itself. To minimize this problem we have changed from a heat seal to the use of a vinyl plastic putty.b In addition, at the time of thawing, the seal on the handle end of the double length straw is cut off prior to This eliminates any pressure in the large air space of the thawing. handle that might cause problems on the embryo side of the straw. The vinyl plug often pops out of the embryo side of the straw during the thaw and relieves pressure on that side. Water contamination is not considered to be a problem if the air space on the plugged end is greater than the space filled with water by capillary action. Because of these problems, thawing instructions should accompany embryo ship-i-tent to avoid preventable loss of embryos upon thawing.

aPres-a-ply

43771,

bCritoseal,

Monoject

JANUARY

Dennison

Manufacturing

Scientific,

1985 VOL. 23 NO. J

St.

Company,

Louis,

Framingham,

Mass.

Mo.

19

THERIOGENOLOGY

3.

Transferring straws to goblets Semen frozen in straws is vapor phase of a large mouth

for storage transferred to goblets for storage in the liquid nitrogen container. However, many of our embryo facilities do not have large mouth liquid nitrogen storage tanks to provide this capability. Even if a large mouth tank is available, we are unlikely to have it on location if we freeze the embryos in an on-farm location. The surface area of a straw is very large for its size and therefore some provisions nust be made to protect the embryos from partial thawing or recrystilization while transferring them to goblets. This is a primary reason for the double length straw. When the embryo is removed from a freezing unit and plunged into liquid nitrogen, This flask is 30 cm. deep and 7 cm. a 1000 ml. dewar flask a is used. wide. The double length straw is 26.5 cm. long and remains upright in the flask after plunging. The embryo containing section of the straw is 12.5 cm. long and a standard storage goblet is 12 cm. deep. Therefore, when the dewar flask is full of liquid nitrogen, the double length straw can be lifted up out of the flask and then lowered into the goblet on a standard cane while the embryo containing portion of the straw stays under the liquid. The goblet is then full of liquid nitrogen when it is transferred into a storage container so that the embryo is always in liquid nitrogen when transferred from one container to another. The large air space in the handle end of the double length straw causes many of them to float when submerged in liquid nitrogen. Therefore, there is a possibility that the straw could float out of a goblet on a cane in a deep storage tank full of liquid nitrogen. To prevent this from happening, one half of a goblet is placed upside down over the straws on the cane as a cap to prevent them from floating out. 4.

Possible contamination by alcohol if an alcohol freezer Unlike semen, which is frozen in the vapor phase of nitrogen tank, several embryo freezing machines utilize an in which the straw is submerged during the freezing process. with faulty seals or a loose wick and powder plug may then However, with the double length to contaminate the embryos. wick and powder plug is in the center of the straw and both sealed minimizing the possibility of contamination by alcohol. FREEZING

is

utilized

a liquid alcohol bath Straws allow alcohol

straw, the ends

are

PROCEDURES

the etiryos are washed and placed Following the embryo collection, groups of 3 or 4 for freezing. The embryos are photographed in using Poloroid film. The embryos are then transferred in to Multi Well Tissue culture plates b and transferred into glycerol and phosphate buffered saline The solutions (Pi3.5) of 0.47 M, 0.94 M and 1.4 M at 10 minute intervals. embryos are then loaded into the labeled double length 112 cc straws and each end is sealed with vinyl plastic putty. into groups

aType bFal

30

no. con

8645, 304 7,

Pope Bet

ton

Scientific

Inc.,

Dickinson

Labware,

Monomonee

Fal

Oxnard,

Ca.

Is,

JANUARY

Wis .

198.5VOL. 23 NO. 1

THERIOGENOLOGY

The sealed straws are then placed into the freezing unit at 20 C and the cooling process is started. From 20 C the temperature is dropped to -7 C at 1.0 C/min. The straws are seeded at -7 C with a hemostat that has been cooled in liquid nitrogen. Seeding is initiated just below the wick and powder plug in the media that was utilized to Once the crystals form in this area wet the powder between the wicks. the process continues beyond the air space in the column of media containing the embryos. The tewerature is held at -7 C for 10 minutes and the occurred.

straws

are

rechecked

to

be

sure

that

crystalization

has

The temperature is droped from -7 C to -35 C at 0.3 Clmin. From The straws are kept at -35 C to -38 C the cooling rate is 0.1 Clmin. -38 C for 15 minutes and then plunged into liquid nitrogen in a dewar flask. are then placed in labeled goblets on canes while in the dewar flask and capped with an inverted 1/2 goblet before being transferred

to

a

nitrogen

tank.

The only change we have made from this protocol has been when using the self-seeding Hoxan Cryoenbryo-PSP Freezer.a Using this freezer we drop from 20 C to -3.0 C at 1 C/min then change to a drop of Instead of 0.1 Clmin during the seeding range of -3.0 C to -7.0 C. checking all straws for evidence of seeding, 1 or 2 dummy straws without embryos are checked to see that seeding has occurred. The remaining freezing rate is the same as for the other freezers. Figure 2

%

TIME FROM COLLECTION TO START OF FREEZING

40

OF R E: C I P 1 1' EK NE TG SN A N T

30

20 59

10

r--

2

.

.

4

6

.

.

.

8

10

12

HOURS

a Hoxan

Corp.,

JANUARY

Sapporo,

Japan

1985 VOL. 23 NO. 1

I

14

I

16

THERIOGENOLOGY Time From collection to Freeze Until late 1982 all embryos were frozen in our home laboratory. In 1982, 50% of our embryo transfer work was done on-farm. This has increased to 75X of our current work being done on-farm. We average 6 donor collections per collection trip and all of our on-farm clients require a travel tine of f to 4 hours. Therefore, embryos collected on-farm for freezing often were held for 10 to 15 hours before being returned to our lab and frozen. When evaluating our pregnancy results we noticed a marked difference in pregnancy rates when donors were collected in-house as corrpared to on-farm. Utilizing data obtained from embryos frozen between July 1, 1982 and June 30, 1984, we saw a 39.0% drop in the overall pregnancy rate based on pregnancies per embryos thawed when embryos were frozen within 4 hours of collection (37.4%) as conpared to 12 hours after collection (22.8%). Figure 2 illustrates this drop in pregnancy rate based on the tine interval from collection to freeze. A decision was made in 1982 to freeze on site those embryos collected on-farm. This required a change to portable freezing equipment. &valuation of Freezing Equipment We have utilized four different pieces of equipment to freeze embryos. We have used two units which utiiize liquid nitrogen as the coolant and two units which are refrigeration units utilizing a recirculating alcohol bath in which the embryos are submerged during the freezing process. The in-house units we have used are the Planer P.T.C. R202/200R and the FTS CRF4Ea adapted with a two pen Bmliscribe recorder.b The portable units we have used are the FTS BC-45-laand the Hoxan Cryoembryo PSP. Although many manufactures don't recom-rendit, we have required a chart recorder that records the chatier tewerature during the freezing process. A copy of this chart is filed with the freezing records so it can be considered when the pregnancy results are evaluated. Since it is often months or years between freezing and thawing of embryos, this helps us remember any power failures or hunen errors that may have occurred during the freezing process. Planer P.T.C. R202/200R This unit has a 25 liter liquid nitrogen tank and utilizes mOre than 10 liters of nitrogen for each procedure. The straws are placed on standard canes but the goblets must be cut away to allow for nitrogen gas flow and access for seeding. The canes most be dram7 up out of the chamber to accomodate seeding and we feel this is an awkward procedure. The straws must be taken off the freezing canes after plunging to transfer them onto canes with standard goblets so that they can be

aFTS System, Inc., Stone Ridge, N.Y. bIndustrial Scientific, Inc., Houston, Texas

‘2

JANUARY

1985 VOL. 23 NO. 1

THERIOGENOLOGY transferred from one tank to another with the goblet full of nitrogen. If the nitrogen tank needs to be refilled when the punp is in place, a hair dryer must be used to remove the condensed moisture from the tubing before insertion of the punp back into the tank. This prevents ice formation within the tubing that interferes with normal function of the unit. FTS CRF 4E This progranmeble freezer has two stages in its conpressor system that will allow one to obtain bath teperatures much lower than those required in currently conventional embryo freezing protocols. We have found that it produces freezing rates much smoother than those of the nitrogen injection sys terns when monitoring temperatures with a thermocouple placed inside a dummy straw. The procedure for seeding is very sin@e since it has an open bath. One needs only to raise the straw out of the alcohol enough to expose that portion of media below the wick and powder plug that was By touching this utilized to solidify the powder between the wicks. media with a liquid nitrogen cooled hemostat the media crystalizes and then passes through the air space and seeds the media colurm containing the embryos. Therefore, the embryos never leave the alcohol bath themselves. This freezer lends itself to portability but is quite bulky with 3 different components and a total weight of 120 pounds. Since we fly to n-any of our clients and take 3 to 4 people plus equipment its weight and

bulk

are prohibitive.

FTS K-45-1 This alcohol freezer is manually controlled which requires fairly close observation but still allows one to perform other tasks such as transferring fresh embryos while the freezing procedure is taking place. It only has a one stage conpressor but this still allows controlled terrperatures below those required for currently conventional embryo freezing procedures. It is a one piece self contained unit that Weighs 75 pounds. We have used this unit extensively and it’s only draw back is that it is bulky and where we have to place it in our airplane causes the center of balance to be too far to the rear of the aircraft. Hoxan Cryoenbryo-PSB This nitrogen injection system is very conpact and comes with carrying cases suitable for checking as baQQaQf? on commrcial airlines or as carry-on luggage. The total weight of the unit and the temerature recorder that we have added is 75 pounds. It uses less than

2 liters of liquid nitrogen for a procedure. We have been very leery of automatic seeding units.

after

much This 9 to 12 mn to us with

However,

testing and challenging we feel comfortable with this system. unit requires that the media containing the embryo be within of the sealed end of the straw. This has also been a concern

regard

to possible

contamination

by thaw

water

during

the

process if the vinyl plastic putty,plug blew out. The need for the media colurm containing the embryos to be within 12 m-nof the end can be eliminated by having a small amount of media The temperature of the seeding touching the vinyl plastic putty seal.

thawing

JANUARY

1985 VOL. 23 NO. 1

THERIOGENOLOGY

block

is

below

-20

C and

will

supercool

and

freeze

the

touching

media

the plug before the media containing the embryos reaches its freezing point. Once the freezing point is reached crystalization begins in the media colum, containing the embryos even though the air space is greater than 12 mn. Care must be taken to be sure that the liquid nitrogen introduced into any nitrogen injection sys tern is clean. Most farm semen tanks when agitated contain debris that can occlude the tubing and orifices of the freezing units. Table

GLYCEROL

I

SOLUTIONS

USED

3 Molar 13.52 Molar Glycerol Phosphate Buffered (serum & antibiotics

Stock

2.22 7.78 lo.00

Saline added)

AMT l-3

CONC.

THAWIffi

EMBRYOS

Solution

f .222 mls stock

ml

SAWLE DILUTION

FOR

Glycerol required)

per

ml

of

DILUTIONS AMT

FOR STRAWS

AMT FOR 10 STRAWS

FOR

5 STRAWS

Stock

PBS

Stock

PBS

Stock

PBS

(1)

1.40

M

1.4

1.6

2.33

2.67

5.61

6.39

(2)

1.17

M

1.17

1.83

1.95

3.05

4.68

7.32

(3)

0.94

M

0.94

2.06

1.57

3.43

3.75

8.25

(41

0.70

M

0.70

2.30

1.17

3.83

2.79

9.21

(5)

0.47

M

0.47

2.53

0. 76

4.24

1.89

10.11

(61

0.23

M

0.23

2.77

0.38

4.72

0.93

Il.07

3 mls

E&ryos remain place Frozen nitrogen storage them nitrogen checked sufficient

14

e&ryos

in each in

holding

dilution media

5 mls

for (PBS

10

I for

minutes. After observation.

12 mls final

dilution,

Etiryo Storage and Handling Frozen embryos are stored in the liquid phase of a large liquid tank (M/E A-90001. The charges for freezing embryos includes for up to 2 years to discourage clients from wanting to keep Shipping containers are filled with liquid in their semen tanks. The nitrogen level is when embryos are transferred to them. orior to removino entmos from a shimer to be sure there is liquid nitrogen to fill the goble’t’ containing the straws.

JANUARY

1985 VOL. 23 NO. 1

THERIOGENOLOGY

Thawinq Procedures Canes containing straws of embryos to be thawed are transferred to a dewar flask filled with liquid nitrogen. The labels can then be easily read by lifting the handle end out of the nitrogen while the embryo end is left submerged. The straws are thawed in warm water at 35 C"for 20 seconds. Prior to thawing, the seal on the handle end of the straw is cut off eliminating any internal pressures from that end. The embryos are transferred into a 1.4 M glycerol solution in a multi well tissue culture plate. The embryos are then taken through 6 decreasing dilutions of glycerol (Table 1) at 10 minute intervals and then put into Dulbecco's Phosphate Buffered Saline containing 20% fetal calf serum and antibiotics. The embryos are photographed and loaded into l/4 cc straws for transfer into recipient cows. The embryos are nudered on the photograph and the straws are coded so that each embryo can be traced to a specific recipient cow in evaluating pregnancy results. Records of Freezing and Thawing When embryos are frozen, a freezing record is filled out. This record includes the names and registration nutiers of the Donor and Service Sire, time from collection to freeze, equipment utilized, cryoprotectant and it's final concentration, the cooling rate and how the containers were labeled. Each straw is listed by number and color code with the stage of development and grade of each embryo within the straw. Freezing records are maintained using a hanging file system.a the pocket in the file contains the freezing record, a copy of the embryo collection record, a copy of the temperature chart made during the freezing procedure, and the photos of the embryos. The front of the file has a chart where we record the storage location, the number of canes utilized, the number of straws utilized and the total number of embryos frozen. This chart is then updated if there is a change in location or if embryos are thawed or shipped out. The Ancom system utilizes color coded letters to simplify filing by the clients name. The individual clients files are kept in The top of the file contains chronological order by collection nutier. a label with the client's name, Donor and Service Sire and the collection nuder. In addition, we place a series of colored signals along the top of the file. these signals stick out above the file and each one represents a straw of frozen embryos. The signal colors match the color of the l/4 cc straw used in the handle portion of the double length straw containing the embryos. A prestype number is put on the front of the tab which corresponds to the nurrber of embryos in that straw. If a straw is thawed or shipped out, this signal is removed from the file at the same time the file is updated.

a Form #SO-0845, Ancom and Conlpanies,Lincoln, Neb.

JANUARY

1985 VOL. 23 NO. 1

THERIOGENOLOGY When embryos are taken out of storage and transported farm, the file is taken with the embryos. If embryos are another person for thawing, a copy of the freezing report sent with the embryos. In addition, we send the photographs embryos being shipped along with a diagram of the double

to a clients shipped to is made and of the length straw

and recomnded thawing procedures. Once all of the embryos have been is transferred to another drawer maintained for at least 6 years. per file and provides easy access useful inventory system. Pregnancy

in

thawed or shipped out, the file filing cabinet where it will be This system costs less than one dollar to records as well as providing a the

Results

Post thaw pregnancy rates were evaluated on embryos frozen between There were a total of 1770 embryos July 1, 1982 and June 30, 1984. thawed of which 1538 were transferred and yielded 507 pregnancies. This represents a pregnancy rate of 28.6% based on pregnancies per embryos thawed and 33.0% based on pregnancies per embryos transferred. There were 1720 embryos recovered after thawing of the 1770 There were no differences embryos frozen for a recovery rate of 97.2%. in recovery rates when conparisons were made by stage of development, quality of embryo or length of tin-e from collection to freezing. Embryos were classified as to stage of development using the following definitions: Advanced Morula: An embryo containing more than 16 cells that have coalesced together to form a conpact mass but has not developed a blas tocoele. Early Blastocyst: An embryo that has formed a blastocoele and has thma signet ring. Blastocyst: An embryo which has a blastocoele that has expanded so that it has lost the signet ring appearance but has not expanded enough to completely fill the zona pellucida. Advanced Blastocyst: A blastocyst that has expanded to fill the entire zona pellucida. When evaluating embryos for quality, Grade 1 embryos were considered normal for their respective stages of development with few if any extraneous degenerate cells. Grade 2 embryos had small inperfections such as extraneous degenerate cells or abnormal shape. Grade 3 embryos had more extraneous degenerate cells or abnormal shape but still contained a mass of cells that appeared viable. Since embryos were frozen in groups of 3 or 4, comparisons of embryo quality or stage of development could only be made when all embryos within the straw were the same quality or stage of development. If all the embryos within one straw were early blastocyst but one the results could be included or more had a different guali ty grade, However, the results could not be when conparing all early blastocys ts. included in evaluation of embryo grade or for early blastocysts of a specific grade (ie early blastocyst grade 11. Likewise, if the embryos in one straw were in different stages of develomnt but all the sane grade, the results could be included when evaluating that grade of

embryo but

I6

would be deleted

from any analysis

involving

JANUARY

stage

and grade.

1985 VOL. 23 NO. I

THERIOGENOLOGY

Pregnancy Results

Table II

By

Quality

Nuder Thawed

Grade

From Quality

Embryo

Frozen Prior

Nunber

Nutier Transferred All

Preqnan

Etiryo

1

470

440

(93.6%)

2

315

274

(87.0%) (63.8%)

lTha wed to Freeze

Pregnancy Rate Per t

Thawed

190 70

47

30

a,~. percentage different by

rates Chi-Square

with the analysis

Transferred

Stages

e

3

Embryos

7

e same superscript (PC .001)

40.5%a’b

43.2%C’d

22.3Xa

25.6%’

14.9%b

23.4%’

are b.

(PC

.05)

significantly d,e

(PC

.1)

Blastocysts 1

146

2

18

138 17

--_

3

52

(94.5%)

2

122

3 a, b,c. different

47.1%

-_

____

-___

Blastocysts

81

(94.2%)’

37

43.1%a

45.7%b

105

(86.1%)’

27

24.2%a

25.%%b

4

1

25.0%

25.0%

4 percentage rates by Chi-Square

44.5%

8

--_

86

37.7%

(94.5%)

Early 1

35.7%

with analysis

the

same (P<

superscript .05)

are

significantly

Advanced Morula 1

77

71 (92.2%)

2

84

70 f%3.3%)d

3

30

21 (70.0%)

a,c,d. different

precentage rates by Chi-Square

JANUARY

with analysis

1985 VOL. 13 NO. I

the

d

same (PC

32

41.6%ayb

45.1%’

14

16. 7%a

2O.O%C

5

16. 7%b

23.9%

superscript .05) b.

(PC

are .I)

significantly

27

THERIOGENOLOGY

Table

III

Pregnancy By Stage

Stage of Development

Results Etiryo

of

Nuke r Thawed

From Frozen/Thawed Development Prior

Nut&e Trans

r

Errbryo Advanced

Blastocys

t

49

Blastocyst Early

Blastocyst

Advanced

Morula

a, b. pregnancy different by

rates Chi-Square

Staqe

Blastocys

Blastocyst Early Advanced

t

Morula

Early Advanced a. by

Advanced

t

Morula

pregnancy Chi-Square

Early

28

tocys

Blastocyst Morula

9%a

43.8%

a%b

37.5%

252

237

83

32.5%’

35.0%

290

242

69

23.8%aybyc

28.5%

the analysis

sarm?

42

superscript fP< .05)

1

c.

are significantly (PC .1/

EnWyos

41 138

19

45.2%

46.3%

52

35.7%

37.7%

86

81

37

43.1%

45.7%

77

71

32

41.6%

45.1%

44.5%a

47.1%

27

24.

2%a

25.8%

14

16.

7%

20.0%

2 Embryos 17

112

8

105

84 rates with analysis

42. 35.

18 Bias

21 69

Grade Blastocyst

Rate Per Transferred

184

146 Blastocys

Pregnancy Thawed

193

with

t

r

Only

48

Grade Advanced

Nurrbe Preg

Errbryos to Freeze

the (P<

70 safre .l)

superscript

Grade

3 Etiryos

4 30

4 21

are

significantly

different

1

25.0%

25.0%

5

16.7%

23.9%

JANUARY

198SVOL.23 NO.1

THERIOCENOLOGY We still consider that freezing groups of embryos in one straw is a sound practice. However, we have made some mistakes when considering analysis of results. Embryos are now being grouped for freezing so that at least the stage of development or the grade are the same. We now freeze nme straws containing 1 or 2 embryos in them to provide for better post thaw analysis. Table II gives the pregnancy results by prefreeze embryo quailty grade. Previous studies of pregnancy rates related to embryo quality prior to fresh transfers showed significant differences related to embryos quality fl), f21, (3). Therefore, it is not surprising to find that this holds true after the embryos are frozen and subsequently

thawed.

The inperfections and degenerated cells seen in the poor quality embryos along with retarded development are indications of stress on the embryos. If these stressed embryos are then submitted to the additional stresses that occur in freezing and thawing, the total stress may be nwre than they can handle. There is a strong indication that this occurs when we conpare the percentages of thawed embryos that are considered to be of transferrable quality after thawing. Of the embryos thawed 96.6% of the grade one embryos where transferred as compared to 87.0% for grade 2 embryos and 63.6% for grade 3 embryos. When submitted to Chi-Square analysis the difference between grade 1 and grade 3 embryos is statistically significant fP< 0.1). The difference between grade 1 and grade 2 embryos approaches statistical significance (PC 0.2). When we restrict the comparisons to the specific stage of embryo development, a statistical difference (PC .05) between the transfer rates for grade 1 and grade 2 embryos is seen for early blastocysts and advanced morula. Table III gives the pregnancy results by the stage of embryo development prior to freezing. When all grades of embryos are grouped together the advanced morulas had a statistically significant lower pregnancy rate than the more advanced stages of development. However, when embryos of the sams grade are conpared, there is no statistical difference in pregnancy rates which indicates this is an effect of embryo grade and not stage of development. This is conpatable with the findings of a previous survey where 88% of grade 3 embryos were morula or advanced morula and 65% of grade 2 embryos were in these sane stages of development (31. It is noteworthy that there is no indication that any of these stages of development are nore or less likely to be successfully frozen and thawed.

1. Elsden, R.P., Nelson, L.D., and Seidel, G.E., Jr. Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotrophin.

Theriogenology 9:17-26 (1978)

2. Schneider, H.J. Jr., Castleberry, R.S. and Griffin, J.L. Commercial aspects of bovine embryo transfer. Theriogenology 13:73-85 (1980) 3.

Wright, J.M. Non-surgical embryo transfer in cattle: interactions. Theriogenology 15:43-56 (1981)

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