Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotrophin

THERIOGENOLOGY

SUPEROVULATING HORMONE

COWS WITH

AND PREGNANT

R. P. Elsden,

FOLLICLE

MARE'S

L. D. Nelson,

STIMULATING

SERUM GONADOTROPHIN and G. E. Seidel,

Jr.

Animal Reproduction Laboratory Colorado State University Fort Collins, CO 80523

ABSTRACT A total of 511 embryos was recovered non-surgically from nearly 100 superovulated or untreated donors. Superovulation with FSH-LH resulted in more corpora lutea, recovered ova, and pregnancies (Pi.01) than superovulation with PMSG. No differences were observed in numbers of ovulations, embryos recovered, or pregnancies per donor when prostaglandin F2c was given to donors 2 versus 3 days following gonadotrophin treatment. Pregnancy rates of 12, 31, 58, and 63% were obtained from groups of embryos classified morphologically as poor, fair, good, and excellent (Pc.05). Morphologically normal embryos collected and transferred at 5 to 6 days of gestation resulted in more (Pc.05) fetuses (75%) than morphologically normal embryos at 8 to 9 days of gestation (56%),but neither was significantly different from morphologically normal embryos at 6.5 to 7.5 days of gestation (61%). There was no difference (P>.O5) between pregnancy rates when retarded embryos from untreated donors (12%) were compared to retarded embryos from superovulated donors (22%). However, a higher proportion of morphologically normal embryos from untreated donors developed into fetuses (71%) than did morphologically normal embryos from superovulated donors (59%, Pc.05). INTRODUCTION The hormone most commonly used to superovulate cows has been pregnant mare's serum gonadotrophin (PMSG), although much of the earlier The gonadotrophic work was done with pituitary extracts (1, 5, 19). substance in PMSG was first described in 1930 (7). Later it was shown that the gonadotrophic effect of PMSG was similar to that of the pituitary gonadotrophins (1, 5, 7, 17, 33) follicle stimulating hormone (FSH) and luteinizing hormone (LH). All three hormones are glycoproteins; however, PMSG possesses more carbohydrate than FSH or LH (24), which accounts for the long half-life of exogenous PMSG (28). PMSG is more similar in amino acid sequence to the LH molecule than FSH. An investigation comparing PMSG and FSH to produce multiple ovulation for twinning was reported in which purified FSH resulted in more

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THERIOGENOLOGY

satisfactory and predictable responses than PMSG (2). It was concluded that this difference was due to biological potency rather than source of gonadotrophins. Further attempts to produce limited multiple ovulations with PMSG (14, 18, 29, 32) or FSH (1, 33, 35) led to the conclusion that the methods used were extremely variable and unreliable. This variability deterred development of a suitable treatment for producing twins in cattle. In another investigation Laster (16) reported that the ovulation rate of PMSG-treated animals was 1.6 + 1.0 compared to 4.4 + 4.9 for FSH. Therefore FSH might be the treatment of choice for obtaizing large numbers of ova from cows. In a recent study, a mixture of FSH plus 20% LH produced more embryos than PMSG in some experiments, but not in others (30). In 1975, PMSG and horse anterior pituitary extract were compared to superovulate cows and heifers (19). Both gonadotrophins were equally effective in cows, but in the heifers, horse anterior pituitary extract produced more non-ovulatory follicles than did PMSG. More ovulations occurred when cows came into estrus from 4 to 7 days after PMSG injection (14, 27). Fewer ovulations resulted if estrus occurred less than 3 days, or later than 7 days following treatment. The interval from injection to estrus was affected by variability in the luteal phase of the estrous cycle, stage of the estrous cycle at which gonadotrophin was administered, and by the dose of PMSG (14, 32). Long intervals between injection and estrus probably depress fertilization rates by creating an unsuitable environment for the gametes at the time of fertilization. Lysing the corpus luteum with exogenous prostaglandin F2o (PGF2o) has led to less variability in the interval between gonadotrophin administration and estrus (10, 26). Only limited information has been available on the morphological characteristics of bovine ova (6, 11, 12, 15, 23). In 1975, the results of an ovum classification system based on morphological characteristics were published (31). The highly rated embryos resulted in more pregnancies following transfer into suitable recipients. Generally, embryos at the stage of development commensurate with the interval from estrus to recovery had a greater chance of becoming fetuses than retarded embryos. Recently a high correlation was demonstrated between metabolism and morphology of day-10 embryos (25). Embryos with a distinct embryonic disc that had hatched from the zona pellucida and had high levels of glucose consumption and lactate production resulted'in a pregnancy rate of 70.5%; in contrast, embryos with the zona pellucida still intact with low levels of glucose consumption resulted in a pregnancy rate of 26.6%. Possibly,tests of metabolic activity can supplement morphological characterization in predicting the survival of embryos. The age of the embryo at the time of transfer is another factor influencing the pregnancy rate. When embryos were recovered and transferred surgically higher pregnancy rates were obtained 5 to 7 days after estrus than 3 to 2 days after estrus (3, 20, 22). There are no published data comparing pregnancy rates obtained with embryos from untreated versus superovulated donors. The recent development of methods for recovering ova non-surgically (9) have

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made such studies feasible. The experiments presented in this paper were designed to investigate: 1.

PMSG versus FSH-LH for superovulating cattle.

2.

The effect of the administration of PGF2o at 48 and 72 hours after gonadotrophin treatment on the number of embryos produced per donor.

3.

An embryo classification system.

4.

The influence of the age of the embryo on pregnancy rate after transfer.

5.

Comparisons of pregnancy rates after transferring embryos from untreated and superovulated donors. MATERIALS AND METHODS

Embryos were recovered from nearly 100 donors of mixed breeds between 13 months and 12 years of age. Superovulation treatment commenced on day 9, 10, or 11 of the estrous cycle (estrus = day 0). Donors were randomly assigned to receive either 1800 I.U. of PMSG or a total of 32 mg of FSH-LH (Armour), plus PGF2, (9) at either 48 or 72 hours after initiating gonadotrophin treatment. If the response to the first treatment (e.g. FSH-LH) was satisfactory (three or more ovulations per ovary), donors were given the basic dose rate of the alternate gonadotrophin (e.g. PMSG) following two normal estrous cycles. If the response was poor, then the dose rate of the second treatment was increased by 50%. Such increases were distributed equally among treatments. Several donors were superovulated a third time. Embryos were also recovered from the same population of donors without superovulation before, between, and after superovulation treatment. Untreated donors were inseminated once approximately l/2 day after observed standing estrus. Superovulated donors were bred on three occasions, l/2, 1, and 1 l/2 days after estrus was first observed, using two, three, and one ampules or straws of semen, respectively. Ova were collected non-surgically 5 to 9 days post-estrus (9). Modified Dulbecco's phosphate buffered saline (PBS) (34) supplemented with 1% heat-treated steer serum was used to flush the ova from the uterus. Ova were then washed in three solutions of sterile medium and stored in PBS supplemented with 10% steer serum at 37C until transfer into recipients that came into estrus within 1 day of the donor. All embryos with at least four clearly defined blastomeres were transferred, even though some embryos were severely retarded. Embryos were transferred surgically into recipients under general anesthesia and pregnancy rates were determined by rectal palpation 85 to 100 days after transfer.

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Embryos were classified into four groups: excellent, good, fair, and poor. Excellent embryos were judged to be at the normal stage of development at the time of examination (3), they were symmetrical, and blastomeres were polygonal in shape forming a tight mass at the morula stage. Embryos classified as good were similar to excellent embryos but were asymmetrical, contained blastomeres excluded from the main morula mass, or were slightly retarded relative to other embryos recovered from the same donor. Fair embryos were retarded 1 to 2 days in development (3), had spherical rather than polygonal blastomeres at the morula stage, contained blastomeres of varying sizes, had signs of degeneration such as large vesicles in the cells, and/or were darker or lighter than normal. Poor embryos were retarded 2 or more days in development, had indistinct cell membranes, and/or had more severe faults than the fair embryos. Data were analyzed by one-way analyses of variance and x2. RESULTS AND DISCUSSION When the 17 PMSG superovulations in Table 1 were compared with the 40 FSH-LH superovulationswith a single degree of freedom comparison, more corpora lutea, ova, and pregnancies were obtained with FSH-LH (Pc.01). However, there was much variability in responses as shown by the standard deviations in Table 1. Numbers of corpora lutea were estimated by rectal palpation and were known to be incorrect in instances where the number of ova collected exceeded the number of corpora lutea palpated; however, the same method of estimation was used for all treatments. The data in Table 1 include only donors without known reproductive problems at the time of treatment. Lengthening the interval between gonadotrophin and estrus by injecting PGF20 3 rather than 2 days after initiating gonadotrophin treatment did not affect (P>.O5) the mean numbers of corpora lutea, ova, or pregnancies (Table 1). Renard -et al. (25) found no difference between administration of PGF20 1 or 2 days following PMSG. However, PGF20 treatment on the same day as PMSG reduced the ovulation rate but increased the incidence of two estrous periods within a few days. There were no significant differences between the pregnancy rates of recipients receiving retarded (poor to fair) embryos at different intervals after estrus (Table 2). The data in Tables 2, 3 and 4 include all embryos transferred during a 16 month period, even if recovered from donors with a history of infertility. Embryos from infertile donors have resulted in normal pregnancy rates (Bowen et&., unpublished). Pregnancy rates obtained after transfer of 5- to 6-day-old normal (good and excellent) embryos were higher (Pc.05) compared to 8- to Y-day-old embryos. The pregnancy rate after transferring normal 6.5- to 7.5-dayold embryos was intermediate. When all embryos were considered, the pregnancy rates were 61 and 49% for 5- to 6-day-old and 8- to Y-day-old embryos, respectively (Pc.05). These results agree with previous experiments (4) in which the uterine environment of superovulated cows was found to be harmful to embryos. Those embryo transferred from superovulated COWS into oviducts of rabbits developed more normally than embryos left in the donor's uterus. This was further substantiated by

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1978 VOL. 9 NO. 1

9

2

Gonadotrophin

PMSG

16

FSH-LH

(5.6)'

(2.2)c

0.9

9.gb

10. gayb (4.9p

1.9

2.5

0.2

Abnormal

6.7ayb

2.ga

Transferrable

11.7b

6.0a

6.4a

Ovulations

Ova

(2.9>c

1.6a

Pregnancies

'Standard deviation (square root of error term of analysis of variance).

a,bMeans within columns with different superscripts are significantly different (Pc.05, Tukey's Test).

24

FSH-LH

PMSG

No. of cows

Interval to PGF2a (days)

Mean number per donor

Responses of Cows to Four Superovulation Treatments

TABLE 1'

?

2

8

B z!

+I

THERIOGENOLOGY

TABLE 2 Pregnancy Rates Obtained After Transferring Embryos of Different Ages Pregnancy rate (X) Normal embryos

Retarded embryos

Total embryos

5 to 6 days

75a(59)C

29(24)

61a(83)

2ga

6.5 to 7.5 days

61ab(217)

17(29)

56ab(246)

12b

8 to 9 days

56b(151)

19(31)

4gb(182)

17b

Age

Percent retarded

a,b Numbers within columns with different superscripts are significantly different (Pc.05). 'Number of embryos. the fact that the percentage of ova from superovulated donors judged as normal declined from 73.6% on day 5 to only 41.7% on day 9 (8), although in the present experiment the highest percentage of abnormal embryos was at 5 to 6 days (Table 2). The lower non-surgical collection rates on days 5 and 6 (21) compared to days 7 to 9 would also have to be considered to optimize the number of pregnancies per donor. There were no significant differences in the pregnancy rates of recipients receiving retarded embryos collected from superovulated versus untreated donors (Table 3). However, pregnancy rates from transfer of normal embryos from untreated donors were slightly higher (Pc.05) than those from superovulated donors. Exogenous gonadotrophins may damage some of the embryos by producing abnormal steroid patterns and the harmful effects may only become obvious as these embryos age. Also, as the number of ovulations increases, the quality of embryos decreases (31). This may explain the difference in the percent of retarded embryos of 8 percentage points between untreated and superovulated donors (Table 3). There was no significant difference in pregnancy rate between good and excellent embryos, but fair embryos resulted in lower pregnancy rates (Pc.05, Table 4). Poor embryos resulted in lower (Pc.05) pregnancy rates than all of the other classifications. Possibly, differences in metabolism could help determine which fair embryos have a good chance of developing into fetuses. Embryos classified as poor (8% in this study, Table 4) should be discarded, unless they have been recovered from particularly valuable donors.

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TABLE 3 Pregnancy Rates Obtained With Embryos From Untreated and Superovulated Donors

Pregnancy rate (X) Untreated

Superovulated

Normal embryos

71a(69)d

5Vb(358)

Retarded embryos

12= (8)

22' (76)

Percent retarded

10

18

a,b,cPregnancy rates with different superscripts are significantly different (Pi.05). d Number of embryos.

TABLE 4 Pregnancy Rates After Transferring Poor, Fair, Good and Excellent Embryos

Classification

No. of embryos

Percent of total embryos

Poor

42

8

12a

Fair

42

8

31b

Good

152

30

58'

Excellent

275

54

63'

Pregnancy rate (X)

a,b,cPregnancy rates with different superscripts are significantly different (Pc.05). Significant advances have been made in bovine embryo transfer techniques since reviewed by Foote and Onuma (13). However, superovulation, embryo recovery, storage, classification and transfer methodology require more basic investigation to maximize the number of pregnancies obtained per donor. Most of the current recommendations for superovulating cows are based on empirical principles. Basic information on follicular maturation and metabolic requirements of embryos (25) are essential requisites to optimize further advances in embryo transfer.

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THERIOGENOLOGY ACKNOWLEDGEMENTS The THAM salt of prostaglandin F2o was generously provided by the Upjohn Co., Kalamazoo, MI through Dr. James Lauderdale. The experiments were partially supported by Select Sires, Inc., Plains City, Ohio and the Colorado State University Experiment Station through Regional Project W-112. The excellent technical assistance of R. Bowen, R. Carter, L. Case, J. Hasler, N. Homan, J. Nash, S. Seidel, and T. Walker is gratefully acknowledged. REFERENCES 1.

Bellows, R. A., Anderson, D. C. and Short, R. E. (1969). Doseresponse relationships in synchronized beef heifers treated with FSH. J. Anim. Sci. g, 638-644.

2.

Bellows, R. A. and Short, R. E. (1972). Superovulation and multiple births in beef cattle. J. Anim. Sci. 3, Suppl. _1, 67-79.

3.

Betteridge, K. J. (1977). Summary of factors affecting success rates in surgical embryo transfer. In: Embryo Transfer in Farm Animals. Ed. K. J. Betteridge. Canada Dept. of Agric. Monograph 16, 29-31.

4.

Boland, M. P., Crosby, T. F. and Gordon, I. (1976). Induction of twin pregnancy in heifers using a simple non-surgical technique. Proc. 8th Int. Congr. Anim. Reprod. A-I., Krakow 2, 241-243.

5.

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6.

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7.

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8.

du Mesnil du Buisson, F., Renard, J. P. and Levasseur, M. C. (1977). Factors influencing the quality of ova and embryos. In: Embryo Transfer in Farm Animals. Ed. K. J. Betterihge. Canada Dept. of Agric. Monograph 16, 24-26.

9.

Elsden, R. P., Hasler, J. F. and Seidel, G. E., Jr. (1976). Nonsurgical recovery of bovine eggs. Theriogenology 5, 523-532.

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R. P., Lewis, S., Cumming, I. A. and Lawson, R. A. S. Superovulation in the cow following treatment with (1974). J.Reprod. Fert. 36, 455-456 PMSG and prostaglandin Fza. (Abstr.).

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14.

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15.

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An unfertilized F. W. (1935). Rec. _6& 25-26.

tubal ovum

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with

16.

Ovulation, fertility and prenatal mortality Laster, D. B. (1973). J. Reprod. in heifers treated with PMSG or porcine FSH. Fert. 2, 275-282.

17.

Laster, D. B., Turman, E. J., Stephens, D. F. and Renbarger, R. E. Ovulation rate of beef heifers treated with PMS. (1970). J. Anim. Sci. 30, 323 (Abstr.).

18.

Laster, D. B., Turman, E. J., Stephens, D. F. and Renbarger, R. E. (1971). Ovulation rates of beef cows and heifers treated with equine gonadotropin (PMS) and chorionic gonadotropin (HCG). J. Anim. Sci. 33, 443-449.

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20.

(1975). Nelson, L. D., Bowen, R. A. and Seidel, G. E., Jr. J. Anim. Sci. 41, Factors affecting bovine embryo transfer. 371-372 (Abstr.).

21.

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22.

Newcomb, R. and Rowson, L. E. A. (1975). Conception rate after uterine transfer of cow eggs in relation to synchronization of oestrus and age of eggs. J. Reprod. Fert. 63, 539-541.

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27.

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28.

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29.

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30.

Seidel, G. E., Jr., Elsden, R. P., Nelson, L. D. and Bowen, R. A. Superovulation of cattle with pregnant mare's serum gonadotropin and follicle stimulating hormone. E.E.C. Seminar: Control of Reproduction in the Cow. Galway, 1977.

31.

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