Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd

THERIOGENOLOGY

FACTORS AFFECTING SURVIVABILITY OF TRANSFERREDWHOLE AND DEMI-EMBRYOS IN A COMMERCIAL DAIRY HERD C. W. Arave, T. D. Bunch, C. H. Mickelsen and K. Warnick Animal, Dairy and Veterinary Sciences Department Utah State University Logan, UT 84322-4815 Received for publication: October 6, 1986 Accepted: July 6, 1987 ABSTRACT Sixty Holstein donor cows were superovulated and embryos were c o l l e c t e d during a 6-d (27 cows) and a 4-d (33 cows) period approximately 60 d apart. Forty-three donor cows yielded embryos. Ninety-one embryos graded 1 or 2 were s p l i t and transferred to 181 recipient Holsteins. Demi-embryos were graded 2, 2-, 3 and 3- prior to transfer. Pregnancy and calving percentages were similar for all demiembryo grades, averaging 59 and 53% from the two donor groups, respectively. Twin demi-embryo pregnancies averaged 36 and 19% for embryos s p l i t at the compacted morula and b l a s t o c y s t stages, respectively. Twin demi-embryo calvings averaged 30 and 15% for these same groups. Progesterone levels of recipients (of either whole or demi-embryos) of second period donors were measured. Pregnancy rate increased generally with level of progesterone; however, calving percentage was slightly greater for recipients with intermediate levels of progesterone (2-6 ng/ml). Multiparous cow (20) recipients of demiembryos had 45% pregnancy and 40% calving, while nulliparous heifer (161) r e c i p i e n t s averaged 59 and 53% pregnancy and c a l v i n g , respectively. Key words: embryo transfer, embryo splitting, twinning INTRODUCTION The potential for producing identical twins from bisected embryos has been demonstrated in several large domestic species. As early as 1974, Trounson and Moore (I) produced identical twins from dividing 4-, 6- and 7-d sheep embryos. Willadsen (2,3) showed that 1, 2 and 4 blastomeres isolated from 2 through 8-cell sheep embryos could produce identical offspring. He utilized a novel approach whereby blastomeres were placed within a surrogate zona pelluclda, sealed in a plug of agar and then cultured in vivo in ligated sheep oviducts. Utilizing the same technique, Willadsen and Polge (4) produced identical twins in cattle. Acknowledgments The authors s i n c e r e l y a p p r e c i a t e the support of Doug Maddox, and the c a t t l e and records supplied by Ruann and Maddox D a i r i e s , R i v e r d a l e and B u r r e l l , CA. The embryo s p l i t t i n g and t r a n s f e r services expertly performed by Em-Tran, Inc., Elizabethtown, PA.are g r a t e f u l l y acknowledged. S t a t i s t i c a l analysis of the data by J. L. Welters is also much appreciated.

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Identical t r i p l e t s and quadruplets in c a t t l e and sheep also have been produced by similar micromanipulatory procedures (5). The c o m m e r c i a l i z a t i o n of embryo t r a n s f e r has led to the exploitation of micromanipulation methods to increase the reproductive potential of genetically superior females (6-15). As a result, not only has the success rate for producing identical twins improved within the last few years, but techniques for producing genetic equivalents have become simplified. Yet the major limiting factor in producing identical twins is the same inherent problem of transfering whole embryos. Only 60 to 65g of good q u a l i t y bovine embryos upon transfer w i l l r e s u l t in calves at term (16-18). The causes of embryonic loss or fetal wastage is s t i l l largely unknown. Heymen(19) has shown that most losses occur before and during implantation and are directly affected by methods of handling embryos. He also reported that pregnancy rates were reduced whenever serum progesterone level of the recipient was <2.00 or >6.00 ng/ml. MATERIALS AND METHODS Sixty Holstein donor cows ranging in age from 2.5 to 7.5 years were superovulated and embryos were collected during a 6-d (27 cows) and a 4-d (33 cows) period approximately 60 d apart. Donor cows had been cycling normally, had two normal estrous periods 19 to 23 days apart, and had calved at least 60 d prior to starting superovulation. Superovulation began 8 to 14 d after estrous period, and consisted of twice d a i l y injections (every 12 h) of f o l l i c l e stimulating hormone (FSH)a for 4 d. Prostaglandin (Lutalyse and Estrumate) was given concommittantly with the seventh and eighth FSH injections to induce luteolysis. Donors were observed for heat and given GnRH at f i r s t sign of heat to cause a more complete release of o v a . Donors were inseminated at least twice (one unit of semen 12 h and a second unit 24 h a f t e r heat; 13 cows). M o s t of the donors (43 cows) were inseminated with three units, at 12 h, 24 h, and 36 h a f t e r heat, and some (four cows) were bred with four units, one at 12 h, two at 24 h, and one at 36 h a f t e r heat, Donors were flushed 7 d following insemination. Flushing media was reconstituted from a powder using s t e r i l e water and consisted of 1% newborn c a l f serum, 1% p e n i c i l l i n streptomycin; Dulbeccos Phosphate Buffered Saline (D-PBS) with 1000 mg D-Glucose/l, 36 mg sodium pyruvate/l, 5 mg phenol red/l and 0.1 gm calcium chloride/l. ~ulture medium in which embryos were maintained was a commercial productu already in solution and consisted of 105 newborn calf serum, 1% penicillin-streptomycin, D-PBS with 1000 mg D-glucose/l, 36 mg sodium pyruvate/l, and 5 mg phenol r e d / l . Culture media was f i l t e r sterilized (0.2 m) prior to use. All embryos were collected and s p l i t by the method of Williams et al. (13)) between B:00 a.m. and 12:00 p.m. Embryos were c l a s s i f i e d according to a method developed at Colorado State University (20). Embryo quality of those s p l i t ranged from 1 to 2, and embryos were at the Burns-Biotec, Omaha, NE b Gibco Laboratories, Life Technologies, Inc., Grand Island, N.Y. a

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t i g h t morula stage of development. Demi-embryos were packaged individually and transferred within a zona pellucida. Embryo transfer was done on the afternoon of or the early evening of the same day as collection. Embryo transfer was done nonsurgically to all cows and to 96% of heifers (the cervix could not be traversed in six heifers). Animals used were Holsteins in good body condition, they were healthy and actively cycling. Recipientand donor estras synchrony was within ± 12 h. Recipientswere palpated the day of transfer to confirm the presence of a corpora luteum. Embryos were transferred to the ipsilateral uterine horn. Recipients of demi-embryos were predominately (89%) nulliparous Holstein heifers. On any given day as the supply of nulliparous heifers was exhausted multiparous Holstein cows were used. Demi-embryos were reclassified prior to transfer and ranged from 2 to 3in quality grade. Pregnancy was determined by palpation per rectum at approximately 60-d post transfer. Three technicians, veterinarians with hundreds of embryo transfers performed previously in a commercial setting, did the embryo transfer during both periods of t h i s study. Recipients were implanted randomly as the technicians worked their way down a l i n e of animals held in locking stanchions. Embryo transfer services and methods used in this study were provided by Em-Tran, Elizabethtown, PA. During the second collection period a r e l a t i v e l y large number of recipients received either whole (61) or demi-embryos (86). Blood samples were collected from these at the time of transfer. Serum was obtained a f t e r centrifugation and stored frozen u n t i l analyzed. Progesterone levels of serum were measured using routine radioimmunoassay (RIA) procedures. Chi-square tests were made to determine significance of differences among demi-embryo grades, twin pregnancies vs. twin b i r t h s , and progesterone levels (three categories, <2, 2-6, >6 ng/ml) at transfer. Donor and recipient animals were in the herds of Douglas Maddox (commercially known as Ruann Farms, Riverdale, CA, and Maddox Dairies, Inc., Burrell, CA). Recipients were managed in a single herd. RESULTS AND DISCUSSION Five hundred seventy-five ova were obtained from 60 superovulated donors during 10 d of collection. Forty-three donors provided 3?4 embryos. Embryos represented 65% of total ova collected or 6.2 embryos per superovulated donor. There was a wide range in donor response to superovulation. No embryos were recovered from 7 donors while 46 unfertilized ova were recovered from another donor. During the f i r s t collection period, 48 of 104 embryos recovered were s p l i t and transferred to 95 recipients (half of one embryo was lost during s p l i t t i n g ) . During the second collection period, 43 of 270 embryos collected were s p l i t and transferred to 86 recipients. Sixtyone whole embryos were also transferred to 61 recipients. During embryo c o l l e c t i o n , as the number of embryos exceeded available recipients, surplus embryos from both collection periods were frozen.

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TABLE 1.

EFFECT OF EMBRYO STAGE AND GRADE AND DEMI-EMBRYO GRADE ON PREGNANCY AT 60 D AND CALVING PERCENTAGE

STAGE

GRADE

1

COHPACTED NORULA

NO. OF OEMI-EMBRYO NO. OF EMBRYOS GRADE DEMI-EMBRYOS

28

21

68 43 43 100 63

61 43 14 100 54

2 23

32 8 2

50 75 100

47 75 i00

q2

57

55

9 7 13

78 29 62

67 lq 62

2 23

1

0

0

TOTAL AND

30

57

50

MORULA STAGE TOTAL AND

128

59

53

G 1 I 8

50 I00 100 63

50 I00 IO0 63

5 3

20 I00

20 100

3-

EARLY BLASTOCYST

CALVED

41 7 7 1 56

TOTAL AND

]5

PRE6. GO O

2 23 3TOTAL AND

i-

RECIPIENTS RECIPIENTS

2 23

1

q

1-

5

TOTAL AND

2

2 2-

3

2

0

,0

TOTAL AND i

10

qo

qO

11 3 4

45 33 50

45 33 50

10

2 23

3TOTAL AND

I

0

O

19

42

42

1

2

2

9

50

5O

2

1

2

2

50

so

1

5

2 23 TOTAL AND

6 3 1 10

67 67 lO0 70

50 33 100 50

BLASTOCYST STAGE TOTAL AND

53

53

47

181

57

51

BLASTOCYST

EXPANDED

BLASTOCYST

OVERALL TOTAL AND i

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THERIOGENOLOGY

Of the 90 embryos which had a potential of providing monozygous twins a f t e r s p l i t t i n g and transfer, 64 were at the compacted morula stage, 18 were at the early blastocyst stage, 3 were at the blastocyst stage, and 5 were at the expanded blastocyst stage. Demi-embryos were graded from 2 to 3- p r i o r to transfer, The overall pregnancy rate of all transferred demi-embryos was 57% (Table 1). This rate is similar to rates reported for whole embryo transfer. Markette et al. (16) reported that the pregnancy rate of whole embryos transferred to 910 recipients was 54.7%. Others have reported pregnancy rates from fewer numbers of whole transferred embryos to range from 43 to 79% for surgical and 25 to 60% for nonsurgical transfers (5,9,18). Overall calving percentage from demi-embryos was 6% below pregnancy percent (Table 1 ). The highest pregnancy percentage occurred for demiembryos obtained from Grade 1 whole embryos. Forty-nine pregnancies resulted from 78 such demi-embryos for a pregnancy rate of 63%. Demi-embryos from compacted morula resulted in the highest calving percentage even though those from expanded blastocysts had a higher 60-d pregnancy rate (Figure 1). Since there was considerable variation in pregnancy and calving percentage among the grades of demi-embryos of the compacted morula, and few blastocysts were s p l i t , none of the differences were significant (P
80.

~E6ENO [Z]Pl;I[GNANCI~ 60OA~

70.

BIB CALV[0

C~ 60. F-" Z W 50k-I r, 40e,,,-

30. 20"

10-

COMPACTED HORULA FIGURE | .

EARLY BLASTOCYST

BLASTOCYST

EXPANDED BLASTOCYST

EMBRYO STA6E AT SPLITTIN6 AND PRE6NANCY RESULTS.

There was no consistent trend for an increase or decrease in pregnancy or in calving percentage among grades of demi-embryos within grades of whole embryos. A high proportion of demi-embryos in Grade 2 and a few embryos in the poorer grades made meaningful comparisons (as in Figure 2) impossible. Data indicate that there was no e f f e c t on

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pregnancy or c a l v i n g percentage of demi-embryo grade. I t was interesting to note the f i r s t calving to a recipient in t h i s study resulted in twin heifers born to a single demi-embryo. The recipient of the twin demi-embryo gave b i r t h to a heifer. These t r i p l e t s resulted from a Grade 2 compacted morula; both demi-embryos were Grade 3. Of the total twin births, 10 pairs were female and 13 pairs were male. 9080-

LEGEND

e'7~PlI[ON~ClE360 D,4~ m cALvm

70oo 60Z 50~40='* 3020lO-

2 FIGURE 2 .

2-

5

3-

GRADE OF DEflI-EBBRYOS (GRADE 2 BEING THE BEST) AT TRANSFER AND PRE6NANCY RESULTS.

Thirty-six percent of the s p l i t compacted morula resulted in twin pregnancies (recipients of both members of a pair of demi-embryos were pregnant), as compared to the combined pregnancy rate of 19% in the early to expanded blastocyst (Figure 3). Calvings were 30 and 15%, respectively, for these same groups. Basedon our original estimate of a 50% calving rate, the chance that both recipients of paired demiembryos would calve was 25%. The actual calving rate of 26% was very close to expectation. Since these results were obtained on a commercial basis with a relatively large samplepthe predictive value of surgical manipulation of embryos and subsequent transfer is further documented. Williams (13) reported similar results for the occurrence of identical twins from sets of two demi-embryos. Progesterone l e v e l s from the second-period r e c i p i e n t s (serum samples collected at time of embryo transfer) receiving e i t h e r s p l i t or nonsplit embryos were compared to pregnancy and calving rates (Figure 4). Pregnancy rate for r e c i p i e n t s of demi-embryos increased with l e v e l s of progesterone. However, there was e s s e n t i a l l y no d i f f e r e n c e in pregnancy rate when progesterone l e v e l s of r e c i p i e n t s r e c e i v i n g whole embryos exceeded 2 ng/ml. There was a marked disadvantage for an embryo placed in a r e c i p i e n t with less than 2 ng/ml progesterone. None of the demi-embryos and o n l y 14% of whole embryos placed in t h i s group of

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THERIOGENOLOGY

,o.1 LEGEND r n RE~mOES s0 D,L~ mm CtLY~

aO.

f.~ 60MQ. I-4 40-

$020100

COMPACTED MORULA

FIGURE 3.

EARLY BLASTOCYST BLASTOCYST

EXPANDED BLASTOCYST

PREGNANCIES AND TWIN BIRTHS TO DERI'EMBRYOmS BY STAGE OF EMBRYO AT S P L I T T I N G .

TWIN

gOBO.

70,

LEGEND

DEMI* |E2] PREX;N/U~IOE~60 OAYS E~RYOSLmm WHOLE [~-~ PREGNANCIES60 DAYS EMBRY0SLr ~ CALVED

$O. FZ

I.~ 40t,4 30"

r///~ ,///~ "JIl!

20-

"//I/,

10-

< 2 ng/ml FIGURE q .

2 to 6 ng/rnl

> 6 ng/ml

SERUM PROGESTERONE LEVELS AT ERBRYO TRANSFER AS RELATED TO SUBSEOUENT PREGNANCY AND C A L V I N 6 .

recipients survived. There appeared to be a slight advantage to embryo survival for recipients with intermediate levels of progesterone, although these differences were not significant. These results further verify the observation of Horthey et al. (21) who found pregnancy rate

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THERIOGENOLOGY

decreased in recipients with serum progesterone levels either below 2 or above 6 ng/ml. Salgado and Donaldson (22) reported that intravaginal progesterone treatment with PRDS improved pregnancy rates in Holstein cows purchased through a saleyard and in recipients in estrus after the donor. In our study, 8% of the recipients had serum progesterone levels less than 2 ng/ml, 74% 2 to 6 ng/ml and 19% greater than 6 ng/ml. Northey et al. (21) observed that 21% of 94 recipients had serum progesterone levels less than 2 ng/ml, 69% had levels of 2 to 6 ng/ml and I0% had levels that exceeded 6 ng/ml. Twenty of the 181 recipients were multiparous cows and had 45 and 40% pregnancy and calving, respectively. One hundred sixty-one nulliparous heifers averaged 59 and 53% pregnancy and c a l v i n g , respectively. Results support the conclusion of others that nulliparous heifers have a higher pregnancy rate than cows (17,18). This was why demi-embryos were p r e f e r e n t i a l l y transferred to heifers in our study when they were available. These data provide additional evidence that embryo quality prior to s p l i t t i n g may be associated with pregnancy rate, as the difference between Grades I and 2 were similar to the differences found by others (7,9). Calving rate was s l i g h t l y greater (not s i g n i f i c a n t ) for demiembryos obtained from the compacted morula stage than the blastocyst stages. Our result differs from the trend found by Williams et al. (7) for Day-7 collections. Additional data are needed to substantiate the correlation of embryo developmental stage at s p l i t t i n g and calving success. Rate of twin b i r t h s to demi-embryos from compacted morula stage were twice that from blastocyst stages, but small numbers and unequal distributions among the various stages prevented any conclusive trends from appearing. The best predictors of pregnancy in this study appeared to be the level of progesterone in the recipients' blood serum and parity. Virgin heifers with more than 2 ng/ml progesterone were the best recipients. REFERENCES 1.

Trounson, W. O. and Moore, N. W. Attempts to produce identical offspring in the sheep by mechanical d i v i s i o n of the ovum. Aust. J. Biol. Sci. 27:505-510 (1974). I

2.

Willadsen, S. M. A method of culture of micromanipulated sheep embryos and i t s use to produce monozygotic twins. Nature, Lond. 277:298-300 (1979).

3.

Willadsen, S. M. The v i a b i l i t y of early cleavage stages containing half the normal number of blastomeres in the sheep, d. Reprod. F e r t i l . 59:357-363 (1980).

4.

Willadsen, S. M. and Polge, C. Attempts to produce monozygotic quadruplets in cattle by blastomere separation. Vet. Rec. 108:211-

213 (1981).

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5.

Willadsen, S. M. Micromanipulation of embryos of large domestic species. In: Adams, C. E., (ed.). Mammalian Egg Transfer. CRC Press, Boca--q~aton, 1982, pp. 185-210.

6.

Willadsen, S. M., Lehn-Jensen, H., F e h i l l y , C. B. and Newcomb R. The production of monozygotic twins in preselected parentage by micromanipulation of n o n s u r g i c a l l y c o l l e c t e d cow embryos. Theriogenology 15:23-29 (1981).

7.

Williams, T. J., Elsden, R. P. and Seidel, C.~.~ Jr. Pregnancy rates with bisected bovi ne embryos. Theriogenology 22:521-531

(1984). 8.

Baker, R. D. and Shea, B. F. Commercial s p l i t t i n g of bovine embryos. Theriogenology 23:3-12 (1985).

9.

Takeda, T., Hallowell, S. V., McGayley, A. D. and Hasler, J. F. Pregnancy rates with i n t a c t and s p l i t embryos t r a n s f e r r e d surgically and nonsurgically. Theriogenology 25:204 abstr. (1986).

10.

Mertes, P. C. and Bondioli, K. R. Effect of splitting technique on pregnancy rate from half embryos. Theriogenology 23:209 abstr.

( 1985). 11.

Warfield, S. d., Seidel, Jr., G. E. and Elsden, R. P. Transfer of bovine demi-embryos w i t h and w i t h o u t zonae p e l l u c i d a e . Theriogenology 2__5:212 abstr. (1986).

12.

O z i l , d. P., Heyman, Y. and Renard, J. P. Production of monozygotic twins by micromanipulation and cervical transfer in the cow. Vet. Rec. 110:126-127 (1982).

13.

Williams, T. d., Elsden, R. P. and Seidel,G. E , J~. Identical t w i n bovine pregnancies d e r i v e d from b i s e c t e d embryos. Theriogenology 1_]_7:114abstr. (1982).

14.

Lambeth, V. A., Looney, C. R., Voelkel, S. A., Jackson, D. A., H i l l , K. G. and Godke, R. A. Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves. Theriogenology 20:85-95 (1983).

15.

Baker, R. D., Eberhard, B. E., L e f f e l , R. E., Rohde, R. F. and Henschen, T . J . Pregnancy rates following surgical transfer of bovine demi-embryos, lOth International Congress on Animal Reproduction and A r t i f i c i a l Insemination (USA) 10:220-220b (1984).

16.

Markette, K. L., Seidel, G.E., J r . and Elsden, R. P. Estimation of embryonic losses in bovine embryo transfer r e c i p i e n t s from progesterone profiles and returns in est~us Theriogenology 2__3:4562 (1985).

17.

Newcomb, R. Egg recovery and transfer in c a t t l e . In: Adams, C. E., (ed.). Mammalian Egg Transfer. CRC Press, Boca'~-aton, 1981, pp. 81-118.

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18.

Sugie, T., Soma, T., Tsunoda, Y. and Mizouchi, K. Survival rates of the embryo during transfer in farm animals. In: Hafez, E. S. E. and Semm, K. (eds.). In Vitro F e r t i l i z a t i o n an~mbryo Transfer. Alan R. Liss, Inc,~N Y, 1982, pp. 335-342.

19.

Heyman, Y. Factors affecting the survival of whole and halfembryos transferred in cattle. Theriogenology 23:63-65 (1985).

20.

Kuzan, F. Classification of embryos prior to freezing. In: Techniques for Freezing Mammalian Embryos, 1984 Short CouPe Proceedings, Animal Reproduction Laboratory, Colorado State University, 1984, pp. 39-50.

21.

Northey, D. L., Barnes, F. L., Eyestone, W. H. and First, N. L. Relationship of serum progesterone, luteinizing hormone and the incidence of pregnancy in bovine embryo transfer recipients. Theriogenology. 23:214 abstr. (1985).

22.

Salgado, R. and Donaldson, L. E. The e f f e c t of i n t r a v a g i n a l progesterone on pregnancy rates in cows receiving embryo transfer. Theriogenology 21:258 abstr. (1984).

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