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., 2

Elizabethtown, PA

17022

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. Registration 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 1983VOL. 19 NO. 1

83

VC? to iuperovu!atjon treatment than cows of various beef breeds tiolsteins azd co~zs 2-f ether dajry breeds oresefit problems whjch (7). 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 2+Gc? P~::JSX have a minimal lactation perfornance. yj Drugs wised in silperoL.iil; 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. KZSpCnSi

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;; The zajoritj cT data presented
and heifers wei-e entered as embryo doqers in a commercial embryc transfer psog?am. Prior to superovulatfon, cows tieye categorized eizber as reoroductively healthy 33" as having fertii?t;f problems. Ferti lit3 problems included (a) failure to coi?celve foi:owing threx er m5re in33a:nat?on~, ib) chronically cystjc ovaries, (c) adhesiorls of the reproductive tract, Superovulation treatments were cot initiated and (d) uterine infections. had exhibited two periods of before 60 days post-parttim and until tom 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

at ?2-hour

intervals

followifig

inseminated

onset

2 or 3 times with frozen

of estrus.

In some cases

semen

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 modification 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 transEmbryos classified as transferrable were composed ferrable. 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, reductions in sums of squares for the various fixed effects were computed Chi-square and F-statistics calculated for the analyses of variance. 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)

JANUARY 1983VOL. 19 NO. 1

85

THERIOGENOLOGY

Table 1.

EFFECT OF REPRODUCTIVE HEALTH ON FIRST SUPEROVULATORY RESPONSE Reproductive status Healthy Infertile

Item Animals, no. Total ova, no. Mean ova/donor Mean fertilized ova/donor Mean embryos/donor Ova fertilized, % Donor with no ova, % Donor with no embryos, % Embryos transferred, no. Pregnant recipients, %

666 6828 10.3 6.7a 6 4a 66," 1ta 3707 68a

Combined

318 1943 6.1b 2.6

984 8771 8.9 5.4 5.1 61 10

2b4

Eb

51b 604b 58

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 superovulation 1 2

i

2 7

8 9 10

No. of cows 35 35 35 35 35 30 25 22 18 12

Means + S.D. Ova/donor Embryos/donor 10.6+9.2 10.8k7.5 10.8k8.4 12.7k8.9 9.458.7 7.8+7-O lO.Ok6.8 9.8k7.7 9.9k7.8 12.6k10.2

7.8k8.0 7.5~6.6 8.3k7.5 7.3rt7.1 6.fk7.7 3.6k4.4 5.0+5.4 2.8k3.6 4.7-15.6 6.3+10.8

Ova fertilized, % 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 1983VOL. 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 However, >nclusion of data from these animals performed on 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,

JANUARY 1983VOL. 19 NO. 1

87

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 literature, 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 disproportionate 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 refractoriness 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 synchronization, this may be a result of problems with heat detection in infertile cows. Consequently, some infertile cows were probably started on super-

88

JANUARY

1983 VOL. 19 NO. 1

= day 0.

d/Based

9

5.6

10.6 10.6 9.7

8.0

6.1

columns

2:6

6.8 6.9 53

5.3

3.8

-

5ob

67: 67: 57o

67a

6ga

Pc.05.

60; 38: ;:ctl

7

Ova fertilized, H I

are significant,

0.6

3.8 2.0 1.9

1.0

63'

%

lK_--

6gb

151 11.4i7.7 7.5k6.6

9oc 83b 83b 97'

;;ba

57a 5ga 62a 42b

:

Ova recovered, H I

(I) COWS

148 11.218.7 6.7k5.8

12

ON FIRST SUPEROVULATORY

(H) AND INFERTILE

60'

108 9.5k7.9 5.4k5.5

OF HEALTHY

Pc.05).

Embryos/donor H I

RESPONSES

CL.

within

3:l

6.7 6.4 54

14.0

-

OvaTdonor H I

superscripts

78 114 7;

2

0

6gb

102 10.0~8.4 6.526.2

are different,

with 10 or fewer palpable

with different

and older

upon animals

*'Numbers

15 years

3 to 6 years 7 to 10 Years 11 to 14 years

282 224 64

26

cows

28

1st calf heifers

No. animals H I

Virgin heifers

Aqe qroup

superscripts

66b

83 9.6k7.4 6.125.6

______~____ 11

FSH INJECTIONS WERE STARTED

Dayestrou
EFFECT OF AGE ON FIRST SUPEROVULATORY

Numbers with different

Table 4.

b,cl

a'Estrus

7ob

Ova fertilized,

%

47 7.8k8.0 5.2k5.6

8

EFFECT OF DAY OF ESTROUS CYCLE ON WHICH RESPONSES OF HEALTHY COWS

Animals, no. Ova/donor, no. Embryos/donor, no.

Item

Table 3.

%d

THERIOGENOLOGY

Table

5.

EFFECT OF DAILY MILK RESPONSES OF HEALTHY

Item

a,b,c,d/

Numbers

Table

-__ ---MiTE-pFrT-(-k$-____~_ 11-20 Zi-30 31-40 41-50 250

10 9.5 4.7

5 13.4 11.6

27 8.5 4.9

113 11.3 7.1

226 11.0 7.2

154 9.0 5.5

34 10.2 6.2

%

51a

87b

63'

65'

6gd

65e

61'

with

different

6.

superscripts

EFFECT OF TIME SUPEROVULATORY


Animals, no. Ova/donor Embryos/donor Ova fertilized,

90

SUPEROVULATORY

l-10

Item

a,b/

ON FIRST

0

Animals, no. Ova/donor Embryos/donor Ova fertilized,

PRODUCTION COWS

Numbers with ent, P<.O5.

%

are different,

SINCE CALVING ON FIRST RESPONSES OF HEALTHY COWS Days since 91-150

calving 151-300

>301 13 7.1 4.3

178 10.5 6.5

262 10.2 6.4

113 10.7 7.3

65a

65a

71b

different

Pc.05.

superscripts

JANUARY

66a

are differ-

1983 VOL. 19 NO. 1

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 Other embryos recovered tended to be lower than from parous animals. 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).

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1983 VOL. 19 NO. 1

91

171 187 130

Summer (May-July)

Autumn (Aug-Ott) 77

94

80

68

9.qb

9.2b

ll.la

11.2a

4.8

6.2

6.6

6.8

No. total ova I H

6.0

6.4

6.3

6.9

2.3

2.2

2.7

2.3

No. embryos I H

67a

71'

61b

652

I 30 73 37 82 67 30

H

58 80 101 187 161 83

1977 1978 1979 1980 1981 1982

No. animals ___ 9.7 9.1 9.9 10.9 9.7 11.8

6.3 4.2 4.8 6.6 7.0 8.8

Ova/donor I H 6.0 I 6.4 5.9

3.1 1.2 1.3

Embryos/donor H I

66 75 64 62 70 61

67 32 27 47 43 35

Ova fertilized, % H I

5oc

42b

4Zb

35a

Ova fertilized,% H I

YEARLY DIFFERENCES IN FIRST SUPEROVULATORY RESPONSES OF HEALTHY (H) AND INFERTILE (I) COWS

Kex_

Table 8.

a b c/ --?--z-Numbers within columns with different superscripts are significant, Pc.05.

178

Spring (Feb-April)

No. animals H I

SEASONAL DIFFERENCES IN FIRST SUPEROVULATORY RESPONSE OF HEALTHY (H) AND INFERTILE (I) ANIMALS

Winter (Nov-Jan)

Season

Table 7.

8 4

?

9

H hJ

g

2

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).

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TMERIOGENOLOGY

Table 9.

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

Item Animals, no.

5 10

Day of recovery after estrus 6 7 8 202

445

253

9 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

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1983 VOL. 19 NO. 1

THERIOGENOLOGY

REFERENCES A device for nonsurgical Drost, M., Brand, A. and Aarts, M.H. Theriogenology 5:503-507 (1976). recovery of bovine embryos. 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). 4.

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

5.

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

6.

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).

7.

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).

8.

Whittingham, D.G. Survival of mouse thawing. Nature (Lond.) 233:125-126

9.

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

10.

Bowen, R.A., Elsden, R.P. and Seidel, G.E., Jr. Embryo transfer for cows with reproductive problems. J. Am. Vet. Med. Assoc. 172:13031306 (1978).

11.

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

12.

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

13.

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).

14.

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).

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1983 VOL. 19 NO. 1

Association

embryos (1971).

of America.

after

transfer.

freezing

and

95

THERIOGENOLOGY

15.

Gunsett, F.C. and Winch, R.P. Factors affecting Critser, J.K., ova transfer in Limousin, Maine-anjou and Simmental cattle. Theriogenology -11:95 (1979).

16.

Holness, D.H., Hale, D.H. and McCabe, C.T. Ovarian response to pregnant mare serum gonadotropin and prostaglandin F2a in Theriogenology Africander and Meshona cows. -14:375-381 (1980).

17.

Shea, B.F., Hines, D.J., Lightfoot, D.E., Ollis, G.W. and Olson, S.M. The transfer of bovine embryos. In: Egg Transfer in Cattle. Commission of the European Communities, Ed. L.E.A. Rowson. Luxembourg. EUR 5491, p. 145-152 (1976).

18.

Sreenan, J.M. and Beehan, D. Methods of induction of superovulation in the cow and transfer results. In: Egg Transfer in Cattle. Commission of the European Communities, Ed. L.E.A. Rowson. EUR 5491, p. 19-34 (1976). Luxembourg.

19.

Castleberry, Schneider, H.J., Jr., aspects of bovine embryo transfer.

20.

Church, R.B. and Shea, B. Some aspects of bovine embryo transfer. In: Egg Transfer in Cattle. Ed. L.E.A. Rowson. Commission of the European Communities, Luxembourg. EUR 5491, p. 73-86 (1976).

21.

McKellar, L.D. and Randel, R.D. Halley, S.M., Rhodes, R.C., III, Successful superovulation, nonsurgical collection and transfer of Theriogenology embryos from Brahman cows. -12: 97-108 (1979).

22.

Garcia, G.J.K., Seidel, shortened FSH treatment -17:90 (1982).

23.

CompariLooney, C.R., Boutte, B.W., Archbald, L.F. and Godke, R.A. son of once daily and twice daily injections for superovulating beef cattle. Theriogenology -15:13-22 (1981).

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