Superovulatory responses of Holstein cows

THERIOGENOLOGY

SUPEROVULATORY RESPONSES OF HOLSTEIN COWS

J.F. Hasler', A.D. McCauley', E.C. Schermerhorn' and R.H. Foote'

1 Em Tran, Inc., Elizabethtown, PA 17022

2 Department of Animal Science, Cornell University Ithaca, NY 14553

ABSTRACT

Approximately 1000 registered cows and heifers were superovulated one to 10 times. Nonsurgical embryo recoveries were performed on all donors which exhibited estrus. Healthy donors produced more total ova and cleaving embryos and had a higher ovum recovery rate, fertilization rate and pregnancy rate from embryos transferred than did cows classified as infertile. While ovum number was not affected during 10 repeated superovulations, fertilization rate and embryo number decreased. The number of ova recovered from healthy cows was affected by season, and from infertile cows by the day of the estrous cycle on which FSH was started and by the number of days since calving. More ova were recovered1 from infertile cows synchronized with prostaglandins prior to superovulation than following a natural estrous cycle. The number of embryos recovered from infertile cows was affected by age and from healthy COWS

by daily milk production. Fertilization rates in both healthy and infertile cows were affected by age, time since calving, daily milk production, day of cycle FSH was injected and season. There was no effect of the day of recovery on the number of ova or embryos recovered from healthy or infertile cows.

INTRODUCTION

Following the development of reliable, efficient non-surgical embryo recovery techniques in the mid 1970's (1,2,3), the use of embryo transfer has grown rapidly in the North American dairy cattle industry. Registra- tion of Holstein calves produced by embryo transfer has doubled yearly, with an increase from 132 calves that were registered in 1976 to 3535 in 1980 (4). Holstein calves comprised 92% of all breeds of dairy calves produced by embryo transfer according to a recent survey (5).

Maximizing superovulatory techniques has been cited as a priority by a majority of workers in the embryo transfer industry (6). An often encountered perception is that lactating Holstein cows are much less

ACKNOWLEDGMENTS: We are grateful to G.P. Brooke, W.D. Logan, R.A. Rushmer, D.J. Tate, R. Whitaker and J. Parrish for aid in collecting and analyzing the data.

JANUARY 1983 VOL. 19 NO. 1 83

KZSpCnSi VC? to iuperovu!atjon treatment than cows of various beef breeds (7). tiolsteins azd co~zs 2-f ether dajry breeds oresefit problems whjch are some&at djfferent f*o"i those enccmtered when itiperov~~lat'9~ beef cows. In dairy cattle, me trming of suaerov~latiofi must often 5e scheduled so as to have a Pi,j%l effect on inter-caiving interva's and lactation perfornance. Drugs wised in silperoL.iil; y j 2+Gc? P~::JSX have a minimal effect upor, daily milk ~rodtiction and riGlk parsty standards. Embryo transfer services 'jr; da-iry cz+t!e are required 0~ a year-rcmd basis I Cyst:c Cvariar! disease and mastitis coqzilic2te tne task 3f sdperov~lating da:yy cows.

The present study is a ~etrospec tive analysis of the sqerovulatiofis and embryo recove-ies per%med between one ar!d T ‘! tin-es on approximatel;; IOKi Holstetr? cows and heifers. The zajoritj cT data presented <s !i,~ited to first superovulations. The objectives of this paper are r_o preser:t a stat<stical picture of svperovu?ation results in @oTstejrl an;mals relative to a number of parameters, incli3d7;ng: a) reproduciitie hea!th of the donor, b) age of the dor?or, cj time since calving, cij daily miik prod~tisn, ej seasor and f] the effects of repeated sdperovu!atinns.

and heifers wei-e entered as embryo doqers in a commercial embryc transfer psog?am. Prior to superovulatfon, cows tieye categorized eizber as reoro- ductively healthy 33" as having fertii?t;f problems. included (a) failure to

Ferti lit3 problems coi?celve foi:owing threx er m5re in33a:nat?on~,

ib) chronically cystjc ovaries, (c) adhesiorls of the reproductive tract, and (d) uterine infections. Superovulation treatments were cot initiated before 60 days post-parttim and until tom had exhibited two periods of estriis. At the time of embryo recovery a record was made of The age of the donor, calvirig date and daily milk production at the last WI4 test prior to sup"rouulatio~ c > * Eonors were palpated per rectum for the presence of a corpus luteurn (CL) prior to initiating superovulation treatment. First day of treatment (days 8 to :3 of the estrous cycle) %r each donor was picked randomly.

Superovulation treatment for a17 animals consisted of twice daily intramuscular (KM) Snjectiocs of descending doses of porcine foilicle stivclating homone (F.S.H., Eurns-Eiotechj for four or ffve days, starting wfth 4 to 3 mg, for a total dose of 26 to 50 mg. Animals received either 1 mg of Cloprosteno! (Cutter Labs., Inc.) or 50 ~g of prostagl aridi ?l F2E (Llpjohnj OF day 2 or 3 of FSH treatment.

Doriors tiere artificially inseminated 2 or 3 times with frozen semen at ?2-hour intervals followifig onset of estrus. In some cases donors were inseminated when tney failed to clear:y exhibit standing estrus but exhibited sjgns such as vagina? m~;cus djscharge or increased prqsical actis;ity. Approximately 75% of the donors were stiperovu!ated and emtiryo recoveries oerformed at the owner's +ann and 25% were at Em :'ran, Inc, Embryo recoveries were performed on a?l donors which were i?semi~ated and the results included in the data,

84

THERIOGENOLOGY

Donors were palpated per rectum prior to embryo recovery and an estimate was made of the number of CL and unovulated follicles. Embryos were recovered 5 to 10 days after estrus (day 0 = estrus) by a modifica- tion of a previously described technique (2). Using unmodified Foley two-way catheters ranging in size from 12 to 24, each uterine horn was separately irrigated with a total of approximately one liter of modified Dulbecco's phosphate-buffered saline (PBS) containing 1% heat-treated new-born calf serum (8). The PBS was collected in one liter graduated cylinders and allowed to stand for 40 min. It was then siphoned off from the top until 100 ml remained. The remainder was poured into a 50x90 mm evaporating dish and examined at 16 magnifications using a stereo microscope. Unfertilized ova and embryos were located and transferred via pipette to PBS containing 10% heat-inactivated new-born calf serum for storage. Embryos were evaluated at 50 magnifications and held at ambient temperature for 12 to 18 hours prior to transfer into recipient animals. Ova were classified as follows:

Total ova included all fertilized and unfertilized ova collected. Fertilized ova included only ova in which at least one cleavage

division had occurred. Embryos included all fertilized ova which were considered trans-

ferrable. Embryos classified as transferrable were composed of a minimum of 16 intact blastomeres.

All recipients were either Holstein heifers (>95%) or Holstein cows which had been observed in estrus within i 36 hours from the time of estrus of the donor. One embryo per recipient was transferred via a flank incision (9) into the lumen of the middle section of the uterine horn iosilateral to the CL, using a 20 gauge plastic catheter attached to a 1 cc syringe.

For statistical analysis, normal equations were generated for each variable of interest for the completely fixed model which included the mean, the geographical location of transfer, season, year, age of donor, health, days fresh, milk production at last test date, day of cycle on which prostaglandin was administered, whether or not estrus was induced and the day embryos were flushed. By a’ least squares procedure, reduc- tions in sums of squares for the various fixed effects were computed and F-statistics calculated for the analyses of variance. Chi-square statistics were calculated for comparison of various ratios obtained when observations were summed over several subclasses. For simplicity of presentation only a portion of the statistical analyses is included.

RESULTS AND DISCUSSION

All embryo recoveries in this study were accomplished without the use of a cervical expander (2) prior to insertion of the catheter. In 1 out of 961 cows (0.1%) and 2 out of 31 virgin heifers (7%) it was not possible to pass a catheter throuqh the cervical canal without risk of damage to the reproductive tract.

Following the first-known superovulation, healthy donors produced more ova, which was only significant at a level of Pc.10, fertilized ova (Pc.05) and embryos (Pc.05) and had a higher rate of fertilization (Pc.05)


THERIOGENOLOGY

Table 1. EFFECT OF REPRODUCTIVE HEALTH ON FIRST SUPEROVULATORY RESPONSE

Item Reproductive status

Healthy Infertile Combined

Animals, no. 666 Total ova, no. 6828 Mean ova/donor 10.3 Mean fertilized ova/donor 6.7a Mean embryos/donor 6 4a Ova fertilized, % Donor with no ova, %

66,"

Donor with no embryos, % 1ta Embryos transferred, no. 3707 Pregnant recipients, % 68a

318 1943

6.1b 2.6

2b4 Eb 51b

604b 58

984 8771

8.9 5.4 5.1

61 10

43:; 67

a,b/ Values for healthy versus infertile animals with different superscripts were different, Pc.05).

Table 2. RESPONSES OF COWS SUPEROVULATED FIVE OR MORE TIMES

Sequence of No. of Means + S.D. Ova fertilized, superovulation cows Ova/donor Embryos/donor %

1 2

i

2 7 8 9 10

35 10.6+9.2 7.8k8.0 35 10.8k7.5 7.5~6.6 35 10.8k8.4 8.3k7.5 35 12.7k8.9 7.3rt7.1 35 9.458.7 6.fk7.7 30 7.8+7-O 3.6k4.4 25 lO.Ok6.8 5.0+5.4 22 9.8k7.7 2.8k3.6 18 9.9k7.8 4.7-15.6 12 12.6k10.2 6.3+10.8

77a 73;

68;c 68d 5oe 53e 34;

Ze

a,b*cyd,eyf'Values for the percentage of ova fertilized with different superscripts are different, Pc.05.

86 JANUARY 1983 VOL. 19 NO. 1

THERIOGENOLOGY

than donors classified as infertile (Table 1). The rancte of variation was similar in both categories, with healthy donors producing from 0 to 38 embryos and infertile cows from 0 to 34. However, a higher percentage of infertile than healthy cows produced no ova or embryos (Pc.05).

There were 91 healthy animals (14%) that failed to produce recoverable embryos the first time they were superovulated (Table 1) compared with 162 (51%) of the infertile animals (Pc.05). Approximately 50% of the animals that failed on the first attempt were superovulated again and 70% of the healthy and 4Zofthe infertile ones produced recoverable etiryos.

There are incomplete records from the early period of this study on the number of superovulated donors which were not inseminated. During 1980-82, a total of 47/856 (5.5%) of healthy donors and 84/447 (18.8%) infertile donors exhibited no signs of estrus following superovulation and were not inseminated. Consequently, embryo recoveries were not performed on these animals. However, >nclusion of data from these animals increases the distinction between healthy and infertile donors: following the first superovulation embryos were not recovered from approximately 18% of healthy and 60% of infertile animals.

A higher pregnancy rate (Pc.05) was achieved with embryos transferred from healthy donors. This was not related to obvious morphological differences of the embryos.

For reasons of schedulina convenience some healthy donors were synchronized with prostaglandrn (PC) prior to initiation of FSH injections. There were no differences in the number of ova (11.6 vs. 10.2) or embryos (6.2 vs. 6.4) recovered from donors superovulated following a P&induced or natural estrus. However, significantly (Pc.05) more ova (9.0 vs. 5.9) but not embryos (4.1 vs. 2.2) were recovered from infertile cows synchronized with PC, versus those superovulated following a natural estrus. Accurate estrus detection in older, infertile cows is often a problem. Therefore, the increased responses of synchronized cows may have resulted from more accurate timing of the initiation of superovulatory treatment. Other studies have demonstrated similar superovulatory responses from infertile cows (10,11,12). However, previous studies, based on small sample sizes, have not demonstrated a significant difference in pregnancy rate of recipients, based on donor fertility, either in mares (13) or in cattle (10,ll). Once pregnancy has been diagnosed, it was reported that neither fertility status of the donor cow nor embryo morphology was related to abortion rates in recipients (14).

Comparisons among studies on superovulation of healthy cows are difficult because of numerous variables, including possible differences in superovulation regimes, breed differences in sensitivitv to superovulation and differences in embryo recovery efficiency. Comparisons of breeds within the same study indicate differences in responsiveness to gonadotropins (15,16,17,18). In numerous reports, superovulated cows of various beef breeds have produced an average of 6 to 8 embryos (11, 19,20,21), although unexplained small responses have al,so been reported in beef cows (22,23). An average of only 3 to 4 embryos has been recovered from superovulated lactating dairy cows in some studies (24,25,


I-HERIOGENOLOGY

26,27) and 5 to 7 in another (28). Differences in the efficacy of pregnant mares serum gonadotropin (PMSG) versus FSH for superovulation may account for some of the reported differences in responses. FSH has been shown to be clearly superior to PMSG in some studies (29,30) while only slight or no differences were detected in other studies (28,31,32, 33).

Estimates of efficiency of embryo recovery vary widely in the litera- ture, In the present study it was difficult to accurately determine the number of CL via palpation per rectum when many CL were present. In 17% of recoveries the number of ova exceeded the estimated number of CL. In all donors in which 10 or fewer CL were estimated, the recovery rate (total ova/CL) was 75.5%. The recovery rate was significantly higher in healthy (85.2%) than in infertile cows (58.8%, Pc.05).

Five or more superovulations were performed on 35 cows with no decline (P>.O5) in the total number of ova for up to 10 superovulations (Table 2). However, there were significant differences in the number of embryos recovered (Pc.05) as a result of the difference in fertilization rate among the first five superovulations. In addition, there was a decrease in fertilization rate after five superovulations. The data were influenced by the types of cows not represented after five superovulations At the request of cow owners, superovulations were often discontinued on animals which had produced large numbers of embryos, leaving a dispropor- tionate number of animals which exhibited lower than averaqe fertilization rates throughout the sequence of superovulations. In spite of variations in fertilization rate, the ability of cows to repeatedly respond to FSH was consistent throughout the series of 10 superovulations.

In other studies, repeated superovulations have been found to result in decreased responses in cattle (34,35). However, the length of time between superovulations has not been consistent. In the present study, all cows were permitted to have two natural estrous cycles between superovulations. In other studies in which cows have been allowed at least one natural estrus, there was no decline in number of superovulations in some (33,36,37) and a decline in others (35,38). It has been suggested that decreased responses following repeated superovulation are due to refrac- toriness from formation of antibodies against the exogenous gonadotrophins used (39). Although an anti-PMSG has been produced in cattle immunized with PMSG injected with Fruend's adjuvant (40), it did not block superovulation when injected into cattle with PMSG. Furthermore, antibodies to PMSG were not identified in cattle repeatedly injected with PMSG (41), although an earlier paper did report an antigonadotrophic effect (39).

There was no significant effect of the day of the estrous cycle FSH injections were started on superovulation in healthy cows (Table 3). In infertile cows, however, (data not presented) analysis of variance revealed that fewer ova per donor were recovered when superovulatory treatment was initiated on day 8 or day 13 compared to cows started in mid-cycle (Pc.05). As pointed out in the discussion of estrous synchron- ization, this may be a result of problems with heat detection in infertile cows. Consequently, some infertile cows were probably started on super-


Table

3.

EFFECT OF DAY

OF ESTROUS

CYCLE

ON WHICH

FSH

INJE

CT

ION

S WERE

STARTED

ON FIRST

SUPEROVULATORY

RESPONSES

OF HEALTHY

COWS

Item

Dayestrou<m?tarted

___

______~____

8 9

10

11

12

lK_--

Animals, no.

47

83

102

108

148

151

Ova/donor,

no.

7.8k8.0

9.6k7.4

10.0~8.4

9.5k7.9

11.218.7

11.4i7.7

Embryos/donor,

no.

5.2k5.6

6.125.6

6.526.2

5.4k5.5

6.7k5.8

7.5k6.6

Ova

fertilized,

% 7ob

66b

6gb

60'

63'

6gb

a'Estrus =

day

0.

b,cl Numbers with different

superscripts

are

different,

Pc.05).

Table

4.

EFFECT OF AGE

ON FIRST

SUPEROVULATORY

RESPONSES

OF HEALTHY

(H)

AND

INFERTILE

(I)

COWS

No.

animals

OvaTdonor

Embryos/donor

Ova

fertilized,

% Ova

recovered,

%d

Aqe

qroup

H I

H I

H I

H I

H I

Virgin heifers

28

0 6.1

- 3.8

- 6ga

1st

calf heifers

26

2 8.0

14.0

5.3

1.0

67a

7

;;b a

:

cow

s 3

to 6

years

282

78

10.6

6.7

6.8

3.8

67:

60;

9oc

57a

7 to 10 Years

years

224

114

10.6

6.4

6.9

2.0

67:

38:

83b

5ga

11 to 14

64

7;

9.7

54

3:l

53

1.9

57o

83b

62a

15 y

ears and

older

9 5.6

2:6

0.6

5ob

;:c tl

97'

42b

*'Numbers

with different

superscripts

within columns

are

significant,

Pc.05.

d/Based

upon animals

with 10 or fewer

palpable CL.

THERIOGENOLOGY

Table 5. EFFECT OF DAILY MILK PRODUCTION ON FIRST SUPEROVULATORY RESPONSES OF HEALTHY COWS

MiTE-pFrT-(-k$-- -__ ---- ____~_

Item 0 l-10 11-20 Zi-30 31-40 41-50 250

Animals, no. 10 5 27 113 226 154 34 Ova/donor 9.5 13.4 8.5 11.3 11.0 9.0 10.2 Embryos/donor 4.7 11.6 4.9 7.1 7.2 5.5 6.2

Ova fertilized, % 51a 87b 63' 65' 6gd 65e 61'

a,b,c,d/ Numbers with different superscripts are different, Pc.05.

Table 6. EFFECT OF TIME SINCE CALVING ON FIRST SUPEROVULATORY RESPONSES OF HEALTHY COWS

Item Days since calving

<go 91-150 151-300 >301

Animals, no. 178 262 113 13 Ova/donor 10.5 10.2 10.7 7.1 Embryos/donor 6.5 6.4 7.3 4.3

Ova fertilized, % 65a 65a 71b 66a

a,b/ Numbers with different superscripts are different, P<.O5.


THERIOGENOLOGY

ovulatory treatment either earlier than day 8 or later than day 13. A decrease in response was reported (42) when cows superovulated with PMSG were treated on day 14 but there were no differences among days 9 to 13. Response was lower when treatment was started very early in the cycle in heifers (43) and sheep (44). Responses in heifers were not increased by exogenous progesterone whether given early or mid-way through the estrous cycle (43) nor by endogenous estradiol concentrations in heifers (45). It has been suggested that the type of follicle population in the ovary at the time of superovulation determines the response (43).

In contrast to an earlier study (46), there was no significant effect (P>.O5) of age on superovulatory response among either healthy or infertile cows (Table 4). Overall there were remarkably small differences in means of the responses between the ages of 2 and 14 years. In 51 cases in which 20 or more embryos were recovered, all but 2 animals were between 3 and 11 years of age. Fertilization rates were significantly higher in fertile cows and heifers younger than 10 years old and in infertile cows younger than 6 years of age.

In fertile animals with fewer than 10 CL, recovery rates were lower in heifers than in older animals. Therefore, in spite of the fact that virgin heifers, based on palpated CL estimates, exhibited large responses to superovulation with a high fertilization rate, the number of ova and embryos recovered tended to be lower than from parous animals. Other published results differ on whether the magnitude of superovulatory response decreased (47) or had no effect on the recovery rate of ova (17, 32).

In contrast with our study, others, in which old age and reproductive health may have been factors, found superovulated heifers to yield more embryos than cows (48,49). The apparent decrease in response among older cows may be related to the number of follicles which has been shown to decrease with increasing age of the cow (50).

Neither daily milk production (Table 5) nor time since calving (Table 6) affected superovulatory response in healthy cows. However, fertilization rate was affected by milk production and time since calving. Although peak fertilization rates were significantly higher in cows at later stages of lactation and at daily production levels below 50 kg of milk, very successful superovulatory responses and fertilization rates were achieved at peak levels of lactation.

The number of ova and embryos recovered from infertile cows did not differ between dry or lactating-cows (P>.O5). However, fewer ova were recovered from 47 infertile cows less than 150 davs fresh than from 220 cows more than 150 days fresh (2.4 vs. 6.6, P<.O5>. The infertile group consisted primarily of cows diagnosed as having chronic cystic ovarian disease. In a study in which the fertility status of the dairy cows was not specified (24), lactating cows produced more embryos than dry cows. The lack of an influence by the magnitude of milk production is consistent with other studies (51,52).


Tabl

e 7.

SE

ASON

AL DI

FFER

ENCE

S IN

FI

RST

SUPE

ROVU

LATO

RY RE

SPON

SE OF

HE

ALTH

Y (H

) AN

D IN

FERT

ILE

(I)

ANIM

ALS

2

No.

anim

als

No.

tota

l ov

a No

. em

bryo

s Ov

a fe

rtil

ized

,%

g

Seas

on

H I

H I

H I

H I

H hJ

Wint

er (N

ov-J

an)

178

68

11.2

a 6.

8 6.

9 2.

3 65

2 35

a 9

Spri

ng (F

eb-A

pril

) 17

1 80

ll

.la

6.6

6.3

2.7

61b

4Zb

?

Summ

er (M

ay-J

uly)

18

7 94

9.

2b

6.2

6.4

2.2

71'

42b

8 Au

tumn

(A

ug-O

tt)

130

77

9.qb

4.

8 6.

0 2.

3 67

a 5o

c 4

a b

c/

--?-

-z- Num

bers

wi

thin

co

lumn

s wi

th di

ffer

ent

supe

rscr

ipts

ar

e si

gnif

ican

t, Pc

.05.

Tabl

e 8.

YE

ARLY

DI

FFER

ENCE

S IN

FI

RST

SUPE

ROVU

LATO

RY RE

SPON

SES

OF HE

ALTH

Y (H

) AN

D IN

FERT

ILE

(I) CO

WS

No.

anim

als

Ova/

dono

r Em

bryo

s/do

nor

Ova

fert

iliz

ed,

% Ke

x_

H I

___

H I

H I

H I

1977

58

30

9.

7 6.

3 6.

0 3.

1 66

67

19

78

80

73

9.1

4.2

I 6.

4 1.

2 75

32

19

79

101

37

9.9

4.8

5.9

1.3

64

27

1980

18

7 82

10

.9

6.6

62

47

1981

16

1 67

9.

7 7.

0 70

43

19

82

83

30

11.8

8.

8 61

35

TMERIOGENOLOGY

There was an influence of season on the number of ova recovered from healthy donors (Table 7, Pc.05). The highest number of ova were recovered in winter and spring and the lowest in summer and autumn. Fertilization rates, however, were significantly higher in summer than in winter in healthy and infertile cows and were highest in autumn in infertile cows.

Although temperature and photoperiod have been shown to affect reproduction in cattle (53) little has been published regarding the influence of season specifically on superovulation. The data in several studies (7,17,32) suggested that responses vary only slightly, if at all, on a seasonal basis. However, the data in two reports (see reference 7) parallel the present study in which the actual number of ova recovered in winter and spring were higher than in summer or fall.

Responses of donors on a yearly basis were not significantly different (Table 8). However, in a study of this type, there may be influences over time from herds sampled, personnel changes, semen used and many other factors which could have compensating effects.

There was no significant effect of day of embryo recovery on the number of ova or embryos recovered (Table 9). Despite the lack of statistical significance, the low number of ova recovered on day 5 are consistent with the known transport rate of ova from the oviducts to the uterus (27,54).


TMERIOGENOLOGY

Table 9. EFFECT OF DAY OF RECOVERY ON THE NUMBER OF OVA AND EMBRYOS FROM HEALTHY AND INFERTILE ANIMALS

Day of recovery after estrus Item 5 6 7 8 9

Animals, no. 10 202 445 253 62

Ova/donor 3.2 8.0 9.0 9.6 8.3

Embryos/donor 2.0 4.7 4.9 5.7 5.5


THERIOGENOLOGY

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

REFERENCES

Drost, M., Brand, A. and Aarts, M.H. A device for nonsurgical recovery of bovine embryos. Theriogenology 5:503-507 (1976).

Elsden, R.P., Hasler, J.F. and Seidel, G.E., Jr. Non-surgical recovery of bovine eggs. Theriogenology 6:523-532 (1976).

Rowe, R.F., Del Campo, M.R., Eilts, C.L., French, L.R., Winch, R.P. and Ginther, O.J. A single cannula technique for nonsurgical collection of ova from cattle. Theriogenology 6:471-483. (1976).

Robertson, Irma, Holstein-Friesian Association of America. Personal communication (1982).

Aichison, T.E. The genetic contribution of embryo transfer. The Advanced Animal Breeder. Feb 15:4-14 (1982).

Seidel, G.E., Jr. and Seidel, S.M. The embryo transfer industry. In: New Technologies in Animal Breeding. Eds. B.G. Brackett, G.E. Seidel, Jr. and S.M. Seidel, Academic Press, N.Y. (1982).

Betteridge, K.J. Techniques and results obtainable in embryo transfer. In: Embryo Transfer in Farm Animals. Ed. K.J. Betteridge, Canada Dept. of Agric. Monograph 16 (1977).

Whittingham, D.G. Survival of mouse embryos after freezing and thawing. Nature (Lond.) 233:125-126 (1971).

Evans, J.F., Hesseltine, G.R. and Kenney, R.M. Standing paralumbar approach for surgical embryo transfer in cattle. Theriogenology 11: 97 (1979).

-

Bowen, R.A., Elsden, R.P. and Seidel, G.E., Jr. Embryo transfer for cows with reproductive problems. J. Am. Vet. Med. Assoc. 172:1303- 1306 (1978).

Elsden, R.P., Nelson, L.D. and Seidel, G.E., Jr. Embryo transfer in fertile and infertile cows. Theriogenology 11: 17-25 (1979). -

Mapletoft, R.J., Johnson, W.H. and Miller, D.M. Embryo transfer techniques in handling repeat breeding cows. Theriogenology 13: 103 (1980).

-

Imel, K.J., Squires, E.L. and Shideler, R.K. A comparison of reproductive performance of fertile versus infertile donor mares. Theriogenology 15:107 (1981).

King, K.K., Elsden, R.P. and Seidel, G.E., Jr. Bovine embryo transfer pregnancies result in normal calves. 2nd world Congress on Embryo Transfer and In-vitro Fertilization, Annecy, France, p. 96 (1982).


THERIOGENOLOGY

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

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Documents

Superovulatory responses of Holstein cows