Evaluation of cryopreservation techniques for bovine embryos

THERIOGENOLOGY

EVALUATION OF CRYOPRESERVATION TECHNIQUES FOR BOVINE ENBRYOS R.S. Prather.l'a l4.F. Spire 1 and R.R. Schalles* Departments of Surgery and Redicinel and Animal Science and Industry* Kannam State University Nanhattan, KS 66506

Received for publication: Accepted:

September 10, 1086 May 27, 1987

ABSTRACT A total of 228 enbryos was nonsurgically collected from auperovulated COVE and dehydratad in dimethyl eulfoxide (DMSO) or glycerol by a three-step procedure or a (T.I.T.) timed interval titration procedure. Embryos were loaded in straws, frozen by cooling to -6.OoC at l.OoC/min, and seeded, followed by cooling to -300C at 0.3OC/min and to -38oC at O.loC/min. At this time the straws were plunged into liquid nitrogen at -1960C. Embryos were thawed in a 270C or 370C water bath and rehydrated by a six-step, three-step (eucrose) or one-atep (aucroae) procedure. Thie yielded a 2 x 2 x 2 x 3 factorial treatment structure. Survival was baaed on development after 12 h in in vitro culture. The only significant single factor affecting survival waa the initial quality grade of the embryo. Grades 1 and 2 embryo8 eurvived more often than Grade 3 embryo8 (P < 0.05). Uaing DNSO aa the cryoprotectant resulted in better acorea for the poet dehydration to post thawing interval (P = 0.02). For both intervals, poat dehydration to post thawing and post thawing to post rehydration, the previoua quality grade waa significant in determining the aubaequent quality grade (P 4 0.01). At each step of the freeze-thaw process, the embryos became progreeaively less morphologically intact. Key words: cryopreservation, DRSO, glycerol, bovine, embryo INTRODUCTIOK A variety of techniques for cryopreservation of embryos has been developed in numerous laboratories around the world. Different cryoprotectante and combinationa and concentrations of cryoprotectants have been added in single or multiple etepe and the embryoa have been either plunged into liquid nitrogen or frozen in rapid (two steps: rapid Acknowledgments Project supported in part by the Kansas Agricultural Experiuent Station Prolect 0483. "Current address: Department of Zoology, University of Wisconsin-Uadiaon, Nadiaon, WI 53706.

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cooling to a aubxero terperature followed by a holding tiaa at that temperature prior to plunging into liquid nitrogen) or alow procedurea (l-6). Eabryoa have aubsequently been thawed in batha at tenperaturea ranging froa 200C to 370C or in air, and rehydration procedures have been six-, three-, and rore recently, one-step processes (7-111. There ia, therefore, a seeaingly endleaa array of corbinationa from which to chooee. This experiment was designed to analyze several coabinationa of the proceaaea and poaaible influencing factora. KATERIALS AND METHODS A total of 228 bovine embryos'was used to evaluate different sethoda of cryopreaervatfon. These eabryos were collected over 15 ao frou Hereford, Siaaental, Holstein, and Angus cowa at Kanaaa State University. Cows were synchronized with prostaglandin and superovulated with 8 or 10 1n)ectiona of follicle atisulating horrone varying between 40 and 50 ag total doaage. The cowa were bred using two atrawa of frozen seaen at each breeding. Esbryoa were collected fros cowa on either Day 7 or 8 (Day 0 = standing eatrua). The collection procedure was nonsurgical and described elaewhere (12). Iaolated eubryoa were placed in culture nedia (CH) containing phosphate buffered saline, 22% heat-treated fetal calf serus, 1% antibiotic antirycotic (all fror Cibco Laboratories, Grand Ialand, NY1 and aaaigned by atage of developaent to one of 24 treatment groupa (Table 1). Table 1. Experirental deaign Six-atep rehydration

DUSO

Glycerol

One-step Three-step rehydration rehydration

Thaw tenperature (Oc) 27

37

27

37

27

37

Three-atep dehydration

Tll

Tl2

T13

T14

T15

T16

T.I.T.

T21

T22

T23

T24

T25

T26

Three-atep dehydration

T31

T32

T33

T34

T35

T36

T.I.T.

T41

T42

T43

T44

T45

T46

T= aeatgned treatnent nueber. T.I.T. = tired interval titration. Eubryoa were dehydrated within 2 h of collection with 1.4K glycerol or 1.W DKSO. The dehydration procedure waa a three-atep or a T.I.T.

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procedure, both carried out at 37oC. The eabryoa assigned to the threeatep dehydration vere moved through three equally increaaing concentrations of DISO or glycerol to a final concentration of 1.5R or l-41(,respectively, both in CR. A lo-min equilibration period was allowed for each atep. The T.I.T. procedure was accomplished by placing the embryos in 5 ml of CR and adding at lo-min intervals specific arounta of 2.M glycerol in CU or 2.2% DRSO in CR so the concentration increased in three equal increaenta to 1.41 glycerol or 1.51 DRSO, reapactively. The concentrated dehydration media (2.131glycerol and 2.251 DtlSO)was added to the diah in a circular notion with a S-ml ayringe. This mixed the aolutiona and aisulated the three-step procedure described earlier without transferring the embryos through dishes. All dehydrated embryos were allowed 15 rin at the final concentration of freezing media prior to placing in the cooling apparatus. After the dehydration process, the embryos were then graded for stage (late lorula, early blastocyst, blaatocyet, expanded blaatocyat, hatched blaatocyst) and quality (1 = excellent, 2 = good, 3 = fair), then loaded into l/2- or l/4-~1 French atrava. The l/4+1 atrawa contained only the respective freezing medium and were sealed with a pair of halostats. The l/2-ml straws contained three fluids separated by air bubbles: CU ('0.05 Al), freezing media containing the embryo ('0.5 ml), and a 0.5 N aucroae solution in CR (' 0.30 ml). The straws were nealed with a plastic plug aiailar to that used by Renard et al. (10) and Leibo (11). Fluid-filled atrawa vere placed in direct contact with a hemostat situated in the path of the liquid nitrogen (LNGfC) exiting vent in a precooled (2OOC) automatic freezer (Cryo-tied.Rode1 1000, Cryo-Red, It. Clellens,III). After an equilibration period of 5 nin at 200C the embryos vere cooled to -6.OoC at l.OoC/nin, at which time a blast of LNC induced seeding aa described by Raaaip et al. (13). Following nucleation the straws were cooled to -3OOC at O.BoC/min and then to a termination temperature of -380C at O.l" Clmin. At the termination tenperature the straws vere plunged into LNC and stored from 1 to 352 d. Following storage the embryos were thawed in a water bath at 27 or 37oc. The straw8 were immersed and gently agitated until 10 aec after complete decrystallization. After thawing, the embryo0 were graded for quality (1 = excellent, 2 = good, 3 = fair, 4 = poor, 5 = degenerate). Only the cell mass was evaluated: cracks in the zonae pellucida were observed very aeldom and therefore were not considered when asaeaaing quality. The embryos frozen in l/4-ml straws were rehydrated by either a aix- or three-atep procedure. The l/2-ml atrawa were rehydrated by a one-step procedure. All rehydrations vere carried out at 370C. The six-8tep rehydration procedure consisted of moving the erbryoa through a series of culture dishes that had equally reduced concentrations of the corresponding cryoprotectant (1.5R, 1.2511,l.OOR, 0.75n, 0.5on, 0.25n, o.oon Dnso: i.4n. 1.1711, 0.931, 0.701, 0.471.

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0.23H,

O.OO?l

glycerol).

A lo-min equilibration period for each step waa

allowed. The three-step rehydration procedure was similar to that previouely (1,8,9) in which the embryo was moved from the freezing mediur to a culture dirh that contained the initial freezing medium plue O.SH At a lo-min interval the embryoa were moved to a 0.511 aucroae l ucroae. solution in CR, then to CR without sucroae.

described

The one-step eucroee rehydration process for the l/2-ml strawa was similar to the three-atep nucroae rehydration but waa accomplished within the &raw a8 described by Renard et al. (10) and Leibo (11). After thawing, the straw was held on its end and tapped lightly until the bubble separating the freezing media and sucrose solutions rose to the top of the straw. This yielded a rehydration solution containing 0.43U sucrose and either 0.20 glycerol or 0.21 DHSO in CM. The straw was then held in an upright position for 10 rin before the enbryo was tran8ferred to CR. Following grading, 104 of the 228 embryo0 were cultured in CR at 370C in a huuidified atmoclphereof air with final viability asseserent baaed on in vitro development after 12 h of culture aa euggented by Rouyaaou and Chupin (31. Of the remaining 124 erbryoa, 107 were transferred to recipient COWI),13 were loat and 4 were outside the range of early blastocyat to expanded blaatocyet. These 124 eubryoa were eliminated from the final analyaie; however, their intermediate quality graden were available and are included in this report (an overview of the experimental procedure is illustrated in Figure 11. All data were analyzed by analyeie of variance using the General Linear Hodtale procedure of Statistical Analysis System (141. All 3-way and higher order interactiona were considered negligible.

Embryo + Dehydration

I[ 1

interval Titration

Glycerol DMSO

Freeze + Thaw

Figure 1.

Quality Graded ACulture

Experimental Procedure

RESULTS Survival

Quality and stage Graded )

AND

DISCUSSION

Analysis.

In vitro culture of post thawed bovine embryoa wae affected only by the initial quality grade of the embryo. Embryos with Gradea 1 and 2 survived significantly more often than Grade 3 embryos (64.8% and 67.8%

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29.1%). These findinga agree with those of Shea et al. (15) and Kennedy et al. (16), who found that higher quality erbryoa have a greater chance of surviving the freeze-thaw process (Table 2).

VI)

In this study a coapariuon of 1.4H glycerol and 1.511DKSO aa the cryoprotectanta ahowed no significant difference in survival rates (Table 21, although glycerol tended to yield higher survival rates than DKSO. Lehn-Jenaen (17) ehoued that DNSO creates greater pH &reawe than glycerol and this say be part of the reaaon for the lower rate, although pH variation8 alone do not Beer to affect eurvival significantly (18). Bilton and Koore (19) found no difference when they coapared l.ON glycerol to 1.51 DKSO. Bouyaaou and Chupin (3) found 1.4K glycerol warna better cryoprotectant than 1.5K DKSO, whereas Frank8 et al. (20) found no difference. Survival rates obtained through differ (P = 0.591 from thoae procedure (57.2X vs 50.6% survival), Nieaann ban shown that 1.4K glycerol

did

not

the T.I.T. dehydration procedure obtained through the three-step which ahould be expected since can be added in a single etep (21).

Table 2. Leaet squares leans for percentage eabryo survival based on single factora Treataent Quality grade 1 2 3

n

Survival

65 26 13

64.8a 67.8a 29.lb

Cryoprotectant DHSO Glycerol

51 53

49.1 58.7

Dehydration method Timed interval titration Three-atep

50 54

57.2 50.6

Thaw tenperature 27oC 37oc

55 49

50.3 57.5

Rehydration procedure Six-step Three-tatep One-step

37 29 38

65.7 52.4 43.5

a*bKeans with different superscripts differ (P < 0.05).

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Thaw tenperaturma (27oC va 37oC) alao did not significantly differ (Table 2). Kasaip et al. (13) had ailrilarresults, whereas Cacherio et al. (7) found that 2OoC resulted in a higher percentage aurvival than 37oc. However, a direct corpariaon of these studies ia not possible since information on the length of tiee for terperature equilibration after decryataliization is not available. No significant differences were found in embryo survival between the rehydration procedurea (Table 2). However, the six-step tended to yield higher survival rates than the one-step (P = 0.08). It has previoualy been reported that the nucroae gradient (three-step) resulted in higher lurvival rates than the six-step rehydration procedure (1,9,22), while Kaaai et al. (8) found no significant difference. Bielanaki et al. (23) have found that a rehydration method lirilar to the three-step described here resulted in significantly higher survival rates than an incremental removal of the glycerol. Previous atudiea coaparing the three-atep or six-step with the one-step have not been lade. The length of embryo storage did not affect survival (P > 0.10) in this study: this finding agrees with the results of Lyon et al. (24). In these studies, souse embryos were frozen up to 5 yr with no decrease in survival ratea. Kaurer (25) stated that there ray be a donor effect; however no significant donor, sire, or breed differences were found in thia ltudy (P > 0.10). No significant differences were found between the stages of development free early blaatocyat to expanded blaatocyat (P > 0.05). The interaction of stage of development by quality grade indicated that higher quality and rore advanced embryo* survive freeze-thawing more often than lower quality, leas developed embryoa (P t 0.05). The advance of labryoa through developaental ltagea indicates a high degree of inherent viability: thus, developmentally advanced enbryoa would have a leaaer chance of containing cellular degeneration that uould impair freeze-thaw survivability. The interaction of rehydration procedure by thaw tenperature indicate6 that the three-atep rehydration procedure resulted in higher survival rates if the e,nbryoawore first thawed at 270C (P t 0.05). Bath temperatures did not significantly influence the survivability of lubryoa rehydrated by the aix- or one-step procedure. Interval Analysis. The roat significant factor affecting the interval from poat dehydration to poat thawing was the initial quality grade of the eabryo (Tablo 3). The results show that Grade 1 eabryoa have significantly better acorea efter thawing than do Grade 2 or 3 embryos (P = 0.002). Eubryoa frozen in DKSO tended to have better acorea after thawing, thus indicating leas overt cellular damage, than did embryos frozen in

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glycerol (P * 0.02). The thaw bath terperature had no significant effect during the interval from poat dehydration to poat thaw (acorea were 1 through 3 before freezing and 1 through 5 after thawing). Tab10 3.

Quality grade effecta from postdehydration to poatthawing

Factor

n

Cryoprotectant DHSO Glycerol

Leaat aquarea meana of quality grade post thauing -

80 106

2.528 2.9lb

Duality grade poat dehydration 1 125 2 37 3 24

2.22= z.84d

s.ogd

Thaw Temperature 27oC 37oc

2.68 2.76

88 98

avbheann with different superscripta differ (P = 0.02). cedhesns with different euperscripta differ (P = 0.002). Neither cryoprotectant nor rohydration procedure affected (P ) 0.10) the change in quality grade from poat thawing to immediately poat rehydration (Table 4). Grade 1 erbryoa had better acorea than Grade 2 (P = 0.0111, 3, 4, or 5 embryoa (P 4 0.001). Grade 2 embryo6 had better l corea than Grade 3, 4 or 5 embryoa (P < 0.001). Grade 3 embryoa had better acorea than Grade 4 or 5 erbryoa (P = 0.003) and Grade 4 embryoa had hotter acores than Grade 5 embryos (P < 0.01). The interval analyses illustrate that embryos tend to deteriorate aa the freeze-thaw proceaa progreaaea, indicating that every atep of the proceaa tend8 to cause more damage to the morphology of the embryo or that degeneration uas progreaaing from aone initial dauage moat probably due to oaaotic gradient changea encountered during the freeze or thaw. The most important factor affecting freeze-thaw survival appears to be the initial quality of the embryo. The optiaal atage of developsent uaa not fully evaluated for a wide range of devolopuental atagea and thus can be applied only to the narrow range from the early to expanded blastooyst &age, where no cignificant differencea were found. The cryoprotectanta, DWSO and glycerol, appear to be of equal value in protecting the embryo. The moat influential factor affecting the quality grade through the intervala from poat dehydration to post thawing and from post thawing to poat rehydration waa the preceding quality of the embryo. Thia factor indicatea that the higher the initial quality of the embryo is the more likely it will be of high

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quality after the freeze-thaw proce88.

Table 4. Quality grade effect8 from po8t thawing to po8t rehydration

Factor

no

Cryoprotectant DIISO Glycerol Rohydration procedure Six-step Thrum-8tmp One-8tep

Lea& 8qUar88 x8en8 Of quality grade po8t rehydration

104 79

3.10 3.13

81 64 38

3.03 3.08 3.23

44 45 62 26 6

1.82” 2.27a

Quality grade poet thawing

zi.ub 3.69'= 4.68“

arbrc*dneans with different 8uper8cripte differ (P < 0.01). *Threa erbryo8 were lo8t during the rahydration proceduree, thu8 accounting for the difference of 3 e8bryo8 between Table 3 (N=186) and Table 4 (N=183).

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10.

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