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Superovulatory and endocrinological responses of simmental cows treated either with PMSG or hMG or in combination
THERIOGENOLOGY SUPEROVULATORY AND ENDOCRINOLOGICAL RESPONSES OF SIMMENTAL COWS TREATED EITHER WITH PMSG OR hMG OR IN COMBINATION G. Bono, G. Gabai, L. Silvestrelli
and Antonella
Comin
Istituto di Produzione Animale, Universitk di Udine 33010 Pagnacco, Udine, Italy Received for pubblication: Accepted:
September 26, 1990 March 28, 1991
ABSTRACT Variations in the plasma concentration of progesterone, total nonconjugated estrogens and luteinizing hormone (LH) were measured in Italian Simmental cows to compare the effects of different superovulation regimens using pregnant mare serum gonadotrophin (PMSG) and human menopausal gonadotrophin (hMG), administered either singly or in combination. The superovulatory response, expressed in terms of the number of recovered andJor transferable ova, was also examined statistically, The cows were divided into four groups, three of which (A, B, C) received different treatments intended to induce superovulation, while the fourth group (D) was used as the control to evaluate endoctinological changes induced by the treatments. Group A cows (n=17) received a single dose of 3000 IU PMSG on Days 11 to 12 of the estrous cycle. Group B cows (n=lZ) received 1000 IU PMSG on Days 11 to 12, followed by seven sequential injections of decreasing doses of hMG at 8-h intervals, and a total of 297.6 IU of FSH and 297.6 IU of LH. Group C cows(n=24) received nine decreasing injections of hMG and a total of 1575 IU of FSH and 1575 IU of LH, at Days 11 to 12 of estrous cycle. All the treated animals and three controls were administered 20 mg PGF on Days 13 to 14 of estrous cycle to induce luteolysis. The experimental results show ecf”)a an intense steroidogenic response to treatment in Groups A and C which, particularly in Group A, could have prejudiced a normal return to reproductive activity; b) a controlled response in Group B; and c) the highest percentage of transferable ova with the combined PMSG and hMG treatment. Key words: cows, superovulation, PMSG, hMG, hormones INTRODUCTION Embryo transfer is used for improving animal production and is being applied to genetic selection, especially in multiple ovulation and embryo transfer. The technique can aho be used to provide ideal animals for scientific research (1). Present superovulation techniques impede the widespread use of embryo transfer on farms. Variability in the recovery of transferable ova is in part attributable to artificial insemination (AI; 2,3) or to the flushing technique (3), however the principle cause lies in the inability to control variations in the hormonal profile induced by superovulation treatments (3,4). The success of superovulation is significantly influenced by individual Acknowledgments The authors thank Prof L. Reichert Jr., Albany Medical College, for kindly supplying ovine LH (LER 1374A) and NIH-LH-B 10 preparation used for the assay and the antibody production; Prof R. Oberosler, Dr V. De Pollo, Dr A. Gusperti for their assistence; Dr C. Mills for help in preparing the paper; Mr A. Fregolent and Mr G. Lenarduzzi for their precious contribution.
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factors such as the species, breed (5 9), age (lo), day of the cycle on which treatment is administered (1 l), and number of supemvulatory treatments to which an animal has been subjected (1 l- 13). Other aspects such as the type and dosage of gonadotrophin used (14), the purity of the products used (11, 15) and the administration of GnRH (16, 17) or associated Iuteolytic agents all influence the outcome. Pregnant mare serum gonadotrophin, which has been widely used and is economical, has been shown to be ineffective in controlling superovulation (18-20), abberration in follicular development (21-23) and notable ovarian hyperstimulation (24, 25). The negative effects may be due to the long half-life of this particular gonadotrophin (18). Several attempts have been made to reduce the time of activity of PMSG, by administering anti-PMSG antibodies (4, 11,26) or by combining another gonadotrophin such as hMG (25) into the treatment. The present trial compares the effects of different superovulation regimens using PMSG and hMG either singly or in combination in Simmental cows. Plasma variations of progesterone (P4), nonconjugated estrogens and LH were investigated, and the superovulatory responses, as determined by the number of transferable ova recovered, were examined statistically. MATERIALS
AND METHODS
Animals and Treatments Italian Simmental cows (n=56), 6 to 13 yr of age were used in the experimental trial. All the cows underwent a gynaecological examination before the commencement of the study. All the cows were found to be free of disease and malformation of the genital organs, and all had regular estrous cycles. The cows were divided into four groups, three of which (Groups A, B and C) received different treatments to induce superovulation; the fourth (Group D) was used as the control to evaluate the endocrinological changes. The treatments given to the three experimental groups commenced on Days 11to 12 of the estrous cycle (estrus = Day 0). Group A cows (n=17) received a singular intramuscular injection of3000 IU of PMSG (FolligonJntervet), followed 48 h later 20 mg of prostaglandin F, (PGF a; Sincrobovis, Gellini), to induce estrus. Group B cows (n=12) received an intramuscular injection of 1000 IU of PMSG, followed by seven injections of hMG (Pergovet 500, Serono) in decreasing doses of 150,75,37.5,18.7,9.4,4.7 and 2.3 IU of LH and FSH. The fit of these seven doses was administered 12 h after the PMSG injection, while the remaining six doses were all given 8 h apart, 48 h after the treatment commenced, 20 mg of PGF, was given. Group C cows (n=24) received nine intramuscular injections ofhMG in decreasing doses, 12 h apart, totalling 1575 IU of LH and 1575 IU of FSH (300,300, 225,225,150,150,75,75 and 75 IU of LH and FSH); 60 h after the commencement of treatment, 20 mg of PGF, were given. Group D (n=3) received 20 mg of PGFza on Days 13 to 14 of the estrous cycle. The three treated groups of cows were artificially inseminated three times, at 8,16 and 24 h after the commencement of estrus, and 7 to 8 dafter the induced estrus, ova were collected by flushing the uterine horns and were then graded. The estrus was clinically detected. Blood samples were taken from four animals in each treatment group, from the jugular vein at 8-h intervals, beginning 48 h before PGF2, administration and ending 88 h after. Blood samples were also taken from the three control animals at 8-h intervals, beginning at the time of PGF, administration until the termination of the experiment.
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The blood was collected in heparinized test tubes, centrifuged at 2000 g for 20 min, and the plasma thus obtained was divided into aliquots and frozen at -20°C until assayed. Plasma Estrogen Assays Evaluation of total nonconjugatedestrogens was carriedout using a slightly modified RIA method described by Seren et al. (27), by extracting 4 ml of plasma with 8 ml of ethylic ether. The antiserum was raised in a rabbit to estriol-16,17- disuccinate-BSA and was used at a dilution of 1:30,000. The following cross-reactions were recorded: estradiol-17P lOO%, estrone92.5%, estriol58.1%, estradiol-17a 51.5%. The sensitivity of the assay, defined as the amount of hormone required to suppress the binding of the labeled hormone to 90% of the binding achieved with no hormone added (B/BO), was 1.8 pg/tube for estradiol-17P. Recovery was 66.8 f 1.5.% for (2,4,6,7-16-17-3H) estradiol-17B. The blank value obtained by extracting under the same conditions an equal amount of double-distilled water was 3.2 f 0.33 pg/ml. Plasma Progesterone Assay Evaluation of progesterone (P4) was carried out using the RIA method described by Seren et al. (27). The antiserum was raised in a rabbit to 1 la-hydroxyprogesteronehemisuccinate-BSA and used at a dilution of 1:5,000. The following cross-reactions were recorded: progesterone lOO%, 1 la-hydroxyprogesterone 77%, 11B-hydxoxyprogesterone 65%, 17a-hydroxyprogesterone 2.9%, 20a-hydroxyprogesterone 0.01% and 20P-hydroxyprogesterone 4.001%. The sensitivity of the assay was 5.4 pgltube. Recovery was 84.8 f 1.2% for (1,2,6,7, 16, 17 -3H) progesterone. The average blank value was < 0.01 rig/ml. Plasma LH Assay An analysis of LH was carried out using the double antibody method as described by Bono et al. (28). The assay required the following reagents: a bovine antiserum ( lmAb) anti-LH, produced in a rabbit, and used at a dilution of 1: 12O,ooO,a purified preparation of ovine LH (LER 1374A), labeled with ‘=I, as described by Salacinski et al. (29), with a specific activity of 25.lpCi/pg;and an anti-rabbit-gammaglobulin serum (2ndAb) produced in a goat and used at a dilution of 1:50. The standard curve was prepared using a highly purified preparation of bovine LH NIH B 10. The sensitivity of the assay was 39.0 pg./tube. The accuracy for all the assays, expressed as percentage of the coefficient of variation, was always less than 15 for the between-assay test and less than 10 for the intra-assay test. Statistical Analysis The data relating to the radioimmunological counts was analyzed with the PRIAMO program (30) mounted on a microcomputer. Both the analysis of variance (ANOVA) and Duncan’s test were used to determine the significance of the differences observed between the experimental groups. The statistical significance of the percentage ova transferability was evaluated using ANOVA and Duncan’s test applied to the value obtained by calculating the arcosine of the square root of the ratio between the number of transplantable ova and the number of ova collected from each cow (angular transformation; 31).
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THERIOGENOLOGY RESULTS Total Estrogens The average variations in estrogen concentrations were found to differ considerably, both between the different groups and among the individuals within the groups (Figure I and Table 1). In each treated group, there was a progressive increase in the average estrogen concentrations, and this increase was the most rapidly noticeable in Group C. Statistically sgnificant differences were observed in the blood estrogen concentrations of the three treated groups (Group C in particular) compared with that of the control group, in the period between the PGF injection and 40 h afterwards. Group B generally had low average values, similar to t%ose of the control group. In Group C, significantly lower estrogen levels were observed between 72 (PcO.01) and 80 h (PcO.05) after PGF, injection. Table 1.
Time (hours) -48 -40 -32 -24 -16 -8 0 8 :: ii 48 56 64
Average (+. SEM) blood concentration of nonconjugated total estrogens (pg/ml) in the four groups of cows. Time 0 indicates the time of PGFza administration Group A (n = 4) 11.12 11.6+ 13.4% 10.5+ 16.9 + 20.3 + 31.8+ 23.5 + 41.0+ 44.8 + 67.9 + 48.3+
0.9 0.7 0.8 1.5 2.9 2.4 8.5 2.9 9.5 13.9 22.4 6.3
ab zb ab cd cd cd ab
44.7+ 7.0 16.1 + 1.6 10.9+ 14.32 1.7 1.6 cd 11.3+ 2.4 ab 15.8 & 2.7
Group B (n = 4)
Group C (n = 4)
7.3 + 1.4 9.3 + 0.2 11.9k2.4 8.9+ 1.4 11.25 1.5 10.3 + 1.9 13.1 + 3.6 13.4k2.0 15.9 + 2.8 23.Ok4.2 25.7 + 7.2 25.854.7 28.2 + 8.1 15.8 + 1.2 11.4 + 0.8 13.7 + 2.7 12.2 + 4.2 14.8 + 6.7
8.4+ 3.0 lO.O+ 0.2 14.7 + 5.2 16.8 * 3.5 14.2+ 2.0 16.2+ 2.3 37.2 + 11.4 47.5 * 15.3 69.0+ 15.8 76.7 + 13.7 106.02 7.8 81.0226.9 63.9 + 28.0 53.3 + 41.9 29.5 + 20.5 7.5+ 1.9 4.5 + 1.2 5.2+ 0.1
a z c” ; a cd ab
Group D (n =3)
b ab b b d d d b
c a
9.6+ 1.6 12.9 + 0.6 13.3 + 1.8 11.1+0.9 11.5kO.8 17.7 22.7 14.220.6 15.3 + 2.7 18.0 + 2.4 17.7k2.0 16.2 + 3.3 13.3 + 1.5
a E ; a d b
El Means with different superscripts on the same coloumn statistically differ at P
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THERIOGENOLOGY
120.
105.
Group A
90.
(3000 IU PMSG)
75. 60. 45. 30. 15.,yfffy-y 0 -46 -32 -16 120 1 105.
0
16
32
40
64
60
Group B
90.
=
E
-
60.
g
45.
;
:;j&r
E
12OJ -46
2
105. 90.
:
75.
L b-
(loo0 IU PMSG + hMG: 297 NJLH and 297 IU FSH given in SCVC~ decreasing dosages)
75.
-32
-16
0
16
32
46
64
60
Group C (hMG:1575 IU LHand 1575 IUFSH given in nine decreasing dosages)
60. 45. 30. 15. 0 -46 120 1 105.
-32
-16
0
16
32
46
64
60
Group D
so.
(Control)
75. 60.
-46
-32
-16
0
16
32
46
64
60
Hours from PGF,Admlnistration
Figure 1. Variations in the blood concentrations (if SEM) of total nonconjugated estrogens in the four groups of cows. Time 0 indicates the time of PGFzcl administration.
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THERIOGENOLOGY
20. 18 _ 16.
Group A (3000IU PMSG)
14 _ 12. 10. 8. 6. 4. 2. 0 -46 202
-32
-16
0
32
46
64
80
18. 16.
Group B (1000IU PMSG
+ hMG: 297 IU LH and297 IUFSHgiven in seven decmsing dosages)
14. 12 _ IO.
\
8. 6. rE
4. 2.
E
O~~.,~..,........., -48 -32 -16 0 20,
* A
18.
16
32
48
64
80
16 -
Group C
14.
(hMGA575 IU LH and 1575 IU FSH given in nine decreasing dosages)
12. 10 8. 6. 4.
-43
-32
-16
0
16
32
48
64
20.1 18 16 i
Group D
14 12. IO _ 8. 6. 4. 2. -48
-32
-16
0
16
32
48
64
80
Hours from PGF,Admlnlstralion
Figure 2.
1184
Variations in the blood concentrations (Z f SEM) of LH in the four groups cows. Time 0 indicates the time of PGF, admiiistration.
Of
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the average P4 concentration remained at 1.4M.9 @ml. The average P4 concentration remained below 1 ng/ml until the end of the experimental period (88 h) in all four groups, even though three cases of statistically significant differences were recorded 32, 56 (P < 0.05) and 88 h (P < 0.01) after the PGF, injection, in which Group C had the highest levels. LH The average LH basal concentrations were found to be generally between 0.7 f 0.2 and 1.1 f 2.0 ng/ml in Group A, 0.62 f 0.1 and 1.1 f 0.2 n&ml in Group B; 0.6 f 0.2 and 1.4 f 0.5 @/ml in Group C; and 0.2 f 0.1 and 0.9 f 0.1 in Group D (Figure 2). A significant difference (PcO.01) between the LH basal concentrations of Group A (0.9 f 0.1 @ml) and Group D (0.3 f 0.1 ng/ml) was seen only 88 h after PGF,, adminstration.The LH preovulatory peaks showed average maximum values 48 h (Group A), 40 h (Group B), values injection, with average 32 h (Group C) and 72 h (Group D) after the PGF of 8.9 f 4.3, 11.6 f 6.3, 13.3 f 12.2, and 7.4 k’P.8 @ml, respectively. The results in Table 2 show the effects of the superovulation treatments on the appearance of the estrogens peak and LH preovulatory peak, and on AI times. In the three treated groups, the estrogens and LH peaks and time of AI were significantly advanced (P&01, PcO.01 and kO.05, respectively) with respect to the control group. The LH peaks occurred after the estrogens peaks at intervals of 2 to 8 h, and they were of reduced amplitude due to the low frequency of blood sampling. Table 2.
Time intervals (hours) between PGF administration estrogens and LH peak and the first AT
Groups A B D C
Estrogens (hours) 38.0 + 3.8 a 42.7 + 5.3 a 69.3 34.0 + 5.3 3.8 ;
(l!Zrs) 46.0 + 5.0 a 44.0+ 2.3 a 41.0+5.2 72.0 + 4.6 6
and the appearance of
First AI (hours) 63.0 + 3.0 ’ 60.0 + 4.9 c 54.0 80.0 + 4.0 3.5 ;
: i Means with different superscripts on the same coloumn statistically differ at P < 0.01. Means with different superscripts on the same coloumn statistically differ at P < 0.05. Group A was given 3000 IU PMSG; Group B received 1000 IU PMSG + hMG (297 IU ofLH and 297 IU of FSH given in seven decreasing doses); Group C received hMG (1575 IU of IJ-I and 1575 IU of FSH given in nine decreasing doses); Group D was the control. Ova Collection Ova were collected from the superovulated cows and were evaluated for embryo transfer suitability and classified as morule, blastocysts, degenerate ova or nonfertilized ova. The average number of ova recovered from Group B was significantly different (P&.01) from that of Groups A and C (Table 3). However, Groups A and C had more nontransferable ova than Group B (PcO.05). The ratio between transferable and recovered ova (Table 4), expressed by angular transformation, indicates that the Group B treatment, in which 86.2% of the ova obtained were transferable, was the most efficient (PcO.05).
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THERIOGENOLOGY Table 3. Average number @ SEM) of ova recovered from the three treated groups and their morphological classification Ova classification
Ova recovered
Group A (n = 17) x+SEM 11.6+ 1.4 a
Group B (n = 12) x+SEM 5.5+0.5
Group C (n = 24) x+SEM b
11.9+ 1.3 a
Ova transferable morule blastocytes
5.42 1.1 2.9 + 0.7 2.5 + 0.9
4.7 f. 0.8 1.4kO.4 3.3 + 0.8
7.7 + 1.1 3.1 +0.8 4.6+ 1.0
Ova not trasferable degenerated not fertilized
6.2* 1.0 ; 2.8 +0.7 d 3.4 f 0.9
0.8kO.2 ,” 0.4 + 0.2 0.420.7 c
4.2 + 0.9 * 2 oTo.5 cd 2:2zo.7 cd
t i Means with different superscripts in the same row statistically differ at P < 0.01. Means with different superscripts in the same row statistically differ at P < 0.05. Group A was given 3000 IU PMSG; Group B received 1000 IU PMSG + hMG (297 IU of LH and 297 N of FSH given in seven decreasing doses); Group C was given hMG ( 1575 N of LH and 1575 IU of FSH given in nine decreasing doses); Group D served as control. Table 4. Absolute number and percentage of ova recovered from the three treatment group. Ova classification
Group A (n =17) n %
Group B (n =12) n %
Group C (n =24) % n
Ova recovered
198 100.0
65 100.0
285 100.0
Ova transferable morule blastocystes Ova not trasferable degenerated not fertilized
93 49 44
47.0 a 24.7 22.3 a
56 16 40
86.2 b 24.6 61.6 b
185 75 110
64.9 b 26.3 38.6 ab
105 47 58
53.0 ; 23.7 29.3 b
9 4 5
13.8 a 6.2 a 7.6 a
loo 48 52
35.1 b 16.9 ab 18.2 ab
a b Means with different superscripts in the same row statistically differ at P < 0.05. Group A was given 3000 N PMSG; Group B received 1000 N PMSG + hMG (297 IU of LH and 297 N of FSH given in seven decreasing doses); Group C was given hMG (1575 IU of LH and 1575 N of FSH given in nine decreasing doses); Group D served as control.
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THERIOGENOLOGY DISCUSSION The analysis of the results demonstrates clearly the intense ovarian stimulation induced by the superovulation treatments. The blood concentrations of the estrogens appeared to depend on the dosage of gonadotrophin used, and in the treated groups these steroids had higher average concentrations than in the control group, with highly significant differences for Group C. Blood estrogen concentrations showed the largest variations within Groups A and C. The estrogen concentrations measured in Group C treated with hMG were similar those reported by McGowan et al. (32) but different from that of other authors (18,33) who evaluated the steroid formation by measuring the concentrations of estradiol-17j3. However, it seems that estradiol-17P shows decidedly lower variations with respect to estradiol-17a and estrone; thus, the simultaneous measurement of estradiol- 1701 and estradiol-17 p is a better method for the evaluation of ovarian stimulation by exogenous gonadotrophins (34). Another effect of the intense ovarian stimulation due to exogenous gonadotrophins could be observed in the persistence of slightly higher P4 levels after treatment with PG in Group C, which in some cases were statistically significant. In this phase, the persistence could be due to the formation of atypical luteinous structures in the ovary. The P4 levels recorded in the later phases of the experiment could be associated with a return to function of the corpora lutea. The hormonal peaks in Group B animals occurred within a narrow time interval; perhaps due to a higher degree of the synchronization of ovulation. Basal levels of LH did not show a significant differences within groups except than that observed 88 h after PG treatment between Group A and Group D. This observation cannot easily explained. The analysis of the data referring to the production of ova in the three groups shows some interesting differences. The PMSG + hMG treatment, in which a less intense steroidogenesis was observed, was characterized by a lower ova production and lower variation. The evaluation of the percentage of transferable ova reveals a higher efficiency of superovulation in Group B. As already stated, the higher degree of synchronization in Group B animals would appear to be further confirmed by the prevalence of one of the two phases of embryonic development classified. With reference to the total number of ova recovered, our results are similar to those of other authors (35, 36), given the doses of gonadotrophin used are taken into account. The number of degenerated ova was decidedly lower in Group B than in Groups A and C. This could have been the consequence of the hormonal and physiological status as shown by the P4/esttogen ratio. Furthermore the hypotesis that the hormonal variations due to the superovulatory treatment may adversely affect sper and/or ova transport, thus interfering with fertilization, has to be considered. It was not possible to establish any correlations between treatments and the percentage of infertile ova because of the practical problem of not being able to use the same semen for AI. The treatment with PMSG or with hMG both provoked a large increase of estrogen concentration, although this response was better controlled in the case of hMG. Clinical observations of PMSG treatment revealed increases in ovary volume to the size of an orange, and cows treated with PMSG showed more irregularities in returning to normal cyclic activity, with a number of cases of ovarian cysts (25). For these reasons, PMSG treatment should not be used in cows which are expected to remain in the herd for some time. From an analysis of our results, and considering the low cost of the PMSGRMG treatment (Group B), we recommend it as an alternative to the very expensive treatment using hMG alone.
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14. Saumande, J. and Chupin, D. Induction of superovulationin cyclic heifers: the inhibitory effect of a large doses of PMSG. Theriogenology x:233-247 (1986).
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THERIOGENOLOGY 15. Saumande, J. Superovulation chez le bovins: actualites et perspectives. Proc. 3rd Meeting A.E.T.E., (Lyon) : 97-141 (1987). 16. Wubishet, A., Graves, C.N., Spahr, S.L. and Kesler, D.J. Continuous subcutaneous infusion of follicle stimulating hormone as a method of superovulating dairy COWS. Theriogenology 25:809-812 (1986). 17. Savage, N.C., Howell, W. and Mapletoft, R.J. Superovulation in the cow using estradiol-178 or GnRH in conjunction with FSHp. Theriogenology =:383-394 (1987). 18. Lauria, A., Genazzani, A.R., Oliva , O., Inaudi, P., Cremonesi, F., Monittola, C. and Aureli, G. Clinical and endocrinological investigation on superovulation induced in heifers by human menopausal gonadotrophin. J. Reprod. Fertil. g:219-225 (1982). 19. Bevers, M. M., Dieleman , S.J., Van Tol, H. T. M., Blankenstein, D. M. and Van Den Broek, J. Changes in pulsatile secretion patterns of LH, FSH, progesterone, androstenedione and oestradiol in cows after superovulation with PMSG. J. Reprod. Ferti1.X: 745-754 (1989). 20. Saumande, J. Concentration of luteinizing hormone, oestradiol-17p and progesterone in the plasma of heifers treated to induce superovulation. J. Endocrinol. 84: 425-437 (1980). 21. Dieleman, S. J. and Kruip, Th. A. M. Relation between 3P-hydroxysteroid dehydrogenase activity in bovine preovulatory follicles and steroids present in the follicular induced oestrus. Proc. 9th Int. Congr. Anim. fluid during normal and PMSG/PGF Reprod. and AI, (Madrid) u: 90-93 $?980). 22. Callesen, H., Greve, T. and Hyttel , P. Premature ovulations in superovulated cattle. Theriogenology B: 155-166 (1987). 23. Hyttel, P., Callesen, H. and Greve ,T. Ultrastructural features of preovulatory oocyte maturation in superovulated cattle. J. Reprod. Fert. 3: 645-656 (1986). 24. Parmigiani, E., Soana, S., Pezzoli, G, and Barbacini, S. Trattamenti superovulatori con PMSG e FSH in bovine da latte. Proc. Soc.It.Buiatria xv: 197-204 (1983). 25. Bono, G., Comin, A., Silvestrelli, L., Gusperti, A., Depollo, V. and Oberosler, R. Variazioni ematiche de1 progesterone, degli estrogeni e dell’LH in bovine sottoposte aaattamenti di superovulazione con PMSG edHMG. Proc. Sot. It. Buiatriam:497511 (1985). 26. Monniaux, population
D., Mariana, J.C. and Gibson, W.R. Action of PMSG on follicular in the heifer. J.Reprod.Fertil. a:243-253 (1984).
27. Seren, E., Leopold , A. and Bolelli, G. Peripheral plasma levels of estrogens and progesterone during the bovine oestrous cycle. Arch. Vet. It. a: l-20 (1974). 28. Bono, G., Gaiani, R. and Chiesa, F. 364-68 (1980).
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Bovine LH radioimmunoassay.
Arch. Vet. It.
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THERIOGENOLOGY 29. Salacinski, P.R.B., McLean, C., Sykes, J.E.C., Clement-Jones, V.V. andLorry, P.J. Iodination of proteins, glycoproteins and peptides using a solid phase oxidizing agent, 1,3,4,6-tetrachloro- 3a,6a-diphenil glicoluril (iodogen). Analytical B&hem. 117: 136-146 (1981). 30. Motta, M. and Degli Esposti, A. A computer program for mathematical treatment of data in Radioimmunoassay. Comp. Progr. Biomed. 13:121-129 (1981). 31. Sokal, R. R. and Rohlf, F. S. Assumption of analysis of variance. In: Biometry. W. H. Freeman and Co., San Francisco 1981, pp. 400-453. 32. McGowan, M.R., Braithwaite, M., Jochle, W. and Mapletoft, R.J. Superovulation of beef heifers with Pergonal (hMG): a dose response trial. Theriogenology 24: 173184 (1985). 33. Alcivar, A.A., Maurer, R.R. and Anderson, L.L. Luteinizing hormone, follicle stimulating hormone, progesterone and estradiol-178 secretion in superovulated beef heifers. Pmt. 10th Congr. Anim. Reprod. andA1, (Urbana-Champaign IL)a:303-305 (1984). 34. Saumande, J. and Lopez-Sebastian, A. Changes in plasma concentration of free and conjugated oestrogens in heifers after treatment to induce superovulation and the relationship with number of ovulations. J. Reprod. Fertil. &411-416 (1982). 35. Hendricks, D.M., Hill, J.R., Dickey, J.F. and Lamond, D.R. Plasma hormone levels in beef cows with induced multiple ovulations. J. Reprod. Fertil. 3:225-233 (1973). 36. Alcivar, A.A., Maurer, R.R. and Anderson, L.L . Superovulatory responses in FSH- or Pergonal-treated heifers. Theriogenology a:635642 (1984).
1190
JUNE 1991 VOL. 36 NO. 6
and Antonella
Comin
Istituto di Produzione Animale, Universitk di Udine 33010 Pagnacco, Udine, Italy Received for pubblication: Accepted:
September 26, 1990 March 28, 1991
ABSTRACT Variations in the plasma concentration of progesterone, total nonconjugated estrogens and luteinizing hormone (LH) were measured in Italian Simmental cows to compare the effects of different superovulation regimens using pregnant mare serum gonadotrophin (PMSG) and human menopausal gonadotrophin (hMG), administered either singly or in combination. The superovulatory response, expressed in terms of the number of recovered andJor transferable ova, was also examined statistically, The cows were divided into four groups, three of which (A, B, C) received different treatments intended to induce superovulation, while the fourth group (D) was used as the control to evaluate endoctinological changes induced by the treatments. Group A cows (n=17) received a single dose of 3000 IU PMSG on Days 11 to 12 of the estrous cycle. Group B cows (n=lZ) received 1000 IU PMSG on Days 11 to 12, followed by seven sequential injections of decreasing doses of hMG at 8-h intervals, and a total of 297.6 IU of FSH and 297.6 IU of LH. Group C cows(n=24) received nine decreasing injections of hMG and a total of 1575 IU of FSH and 1575 IU of LH, at Days 11 to 12 of estrous cycle. All the treated animals and three controls were administered 20 mg PGF on Days 13 to 14 of estrous cycle to induce luteolysis. The experimental results show ecf”)a an intense steroidogenic response to treatment in Groups A and C which, particularly in Group A, could have prejudiced a normal return to reproductive activity; b) a controlled response in Group B; and c) the highest percentage of transferable ova with the combined PMSG and hMG treatment. Key words: cows, superovulation, PMSG, hMG, hormones INTRODUCTION Embryo transfer is used for improving animal production and is being applied to genetic selection, especially in multiple ovulation and embryo transfer. The technique can aho be used to provide ideal animals for scientific research (1). Present superovulation techniques impede the widespread use of embryo transfer on farms. Variability in the recovery of transferable ova is in part attributable to artificial insemination (AI; 2,3) or to the flushing technique (3), however the principle cause lies in the inability to control variations in the hormonal profile induced by superovulation treatments (3,4). The success of superovulation is significantly influenced by individual Acknowledgments The authors thank Prof L. Reichert Jr., Albany Medical College, for kindly supplying ovine LH (LER 1374A) and NIH-LH-B 10 preparation used for the assay and the antibody production; Prof R. Oberosler, Dr V. De Pollo, Dr A. Gusperti for their assistence; Dr C. Mills for help in preparing the paper; Mr A. Fregolent and Mr G. Lenarduzzi for their precious contribution.
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factors such as the species, breed (5 9), age (lo), day of the cycle on which treatment is administered (1 l), and number of supemvulatory treatments to which an animal has been subjected (1 l- 13). Other aspects such as the type and dosage of gonadotrophin used (14), the purity of the products used (11, 15) and the administration of GnRH (16, 17) or associated Iuteolytic agents all influence the outcome. Pregnant mare serum gonadotrophin, which has been widely used and is economical, has been shown to be ineffective in controlling superovulation (18-20), abberration in follicular development (21-23) and notable ovarian hyperstimulation (24, 25). The negative effects may be due to the long half-life of this particular gonadotrophin (18). Several attempts have been made to reduce the time of activity of PMSG, by administering anti-PMSG antibodies (4, 11,26) or by combining another gonadotrophin such as hMG (25) into the treatment. The present trial compares the effects of different superovulation regimens using PMSG and hMG either singly or in combination in Simmental cows. Plasma variations of progesterone (P4), nonconjugated estrogens and LH were investigated, and the superovulatory responses, as determined by the number of transferable ova recovered, were examined statistically. MATERIALS
AND METHODS
Animals and Treatments Italian Simmental cows (n=56), 6 to 13 yr of age were used in the experimental trial. All the cows underwent a gynaecological examination before the commencement of the study. All the cows were found to be free of disease and malformation of the genital organs, and all had regular estrous cycles. The cows were divided into four groups, three of which (Groups A, B and C) received different treatments to induce superovulation; the fourth (Group D) was used as the control to evaluate the endocrinological changes. The treatments given to the three experimental groups commenced on Days 11to 12 of the estrous cycle (estrus = Day 0). Group A cows (n=17) received a singular intramuscular injection of3000 IU of PMSG (FolligonJntervet), followed 48 h later 20 mg of prostaglandin F, (PGF a; Sincrobovis, Gellini), to induce estrus. Group B cows (n=12) received an intramuscular injection of 1000 IU of PMSG, followed by seven injections of hMG (Pergovet 500, Serono) in decreasing doses of 150,75,37.5,18.7,9.4,4.7 and 2.3 IU of LH and FSH. The fit of these seven doses was administered 12 h after the PMSG injection, while the remaining six doses were all given 8 h apart, 48 h after the treatment commenced, 20 mg of PGF, was given. Group C cows (n=24) received nine intramuscular injections ofhMG in decreasing doses, 12 h apart, totalling 1575 IU of LH and 1575 IU of FSH (300,300, 225,225,150,150,75,75 and 75 IU of LH and FSH); 60 h after the commencement of treatment, 20 mg of PGF, were given. Group D (n=3) received 20 mg of PGFza on Days 13 to 14 of the estrous cycle. The three treated groups of cows were artificially inseminated three times, at 8,16 and 24 h after the commencement of estrus, and 7 to 8 dafter the induced estrus, ova were collected by flushing the uterine horns and were then graded. The estrus was clinically detected. Blood samples were taken from four animals in each treatment group, from the jugular vein at 8-h intervals, beginning 48 h before PGF2, administration and ending 88 h after. Blood samples were also taken from the three control animals at 8-h intervals, beginning at the time of PGF, administration until the termination of the experiment.
JUNE 1991 VOL. 36 NO. 6
The blood was collected in heparinized test tubes, centrifuged at 2000 g for 20 min, and the plasma thus obtained was divided into aliquots and frozen at -20°C until assayed. Plasma Estrogen Assays Evaluation of total nonconjugatedestrogens was carriedout using a slightly modified RIA method described by Seren et al. (27), by extracting 4 ml of plasma with 8 ml of ethylic ether. The antiserum was raised in a rabbit to estriol-16,17- disuccinate-BSA and was used at a dilution of 1:30,000. The following cross-reactions were recorded: estradiol-17P lOO%, estrone92.5%, estriol58.1%, estradiol-17a 51.5%. The sensitivity of the assay, defined as the amount of hormone required to suppress the binding of the labeled hormone to 90% of the binding achieved with no hormone added (B/BO), was 1.8 pg/tube for estradiol-17P. Recovery was 66.8 f 1.5.% for (2,4,6,7-16-17-3H) estradiol-17B. The blank value obtained by extracting under the same conditions an equal amount of double-distilled water was 3.2 f 0.33 pg/ml. Plasma Progesterone Assay Evaluation of progesterone (P4) was carried out using the RIA method described by Seren et al. (27). The antiserum was raised in a rabbit to 1 la-hydroxyprogesteronehemisuccinate-BSA and used at a dilution of 1:5,000. The following cross-reactions were recorded: progesterone lOO%, 1 la-hydroxyprogesterone 77%, 11B-hydxoxyprogesterone 65%, 17a-hydroxyprogesterone 2.9%, 20a-hydroxyprogesterone 0.01% and 20P-hydroxyprogesterone 4.001%. The sensitivity of the assay was 5.4 pgltube. Recovery was 84.8 f 1.2% for (1,2,6,7, 16, 17 -3H) progesterone. The average blank value was < 0.01 rig/ml. Plasma LH Assay An analysis of LH was carried out using the double antibody method as described by Bono et al. (28). The assay required the following reagents: a bovine antiserum ( lmAb) anti-LH, produced in a rabbit, and used at a dilution of 1: 12O,ooO,a purified preparation of ovine LH (LER 1374A), labeled with ‘=I, as described by Salacinski et al. (29), with a specific activity of 25.lpCi/pg;and an anti-rabbit-gammaglobulin serum (2ndAb) produced in a goat and used at a dilution of 1:50. The standard curve was prepared using a highly purified preparation of bovine LH NIH B 10. The sensitivity of the assay was 39.0 pg./tube. The accuracy for all the assays, expressed as percentage of the coefficient of variation, was always less than 15 for the between-assay test and less than 10 for the intra-assay test. Statistical Analysis The data relating to the radioimmunological counts was analyzed with the PRIAMO program (30) mounted on a microcomputer. Both the analysis of variance (ANOVA) and Duncan’s test were used to determine the significance of the differences observed between the experimental groups. The statistical significance of the percentage ova transferability was evaluated using ANOVA and Duncan’s test applied to the value obtained by calculating the arcosine of the square root of the ratio between the number of transplantable ova and the number of ova collected from each cow (angular transformation; 31).
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THERIOGENOLOGY RESULTS Total Estrogens The average variations in estrogen concentrations were found to differ considerably, both between the different groups and among the individuals within the groups (Figure I and Table 1). In each treated group, there was a progressive increase in the average estrogen concentrations, and this increase was the most rapidly noticeable in Group C. Statistically sgnificant differences were observed in the blood estrogen concentrations of the three treated groups (Group C in particular) compared with that of the control group, in the period between the PGF injection and 40 h afterwards. Group B generally had low average values, similar to t%ose of the control group. In Group C, significantly lower estrogen levels were observed between 72 (PcO.01) and 80 h (PcO.05) after PGF, injection. Table 1.
Time (hours) -48 -40 -32 -24 -16 -8 0 8 :: ii 48 56 64
Average (+. SEM) blood concentration of nonconjugated total estrogens (pg/ml) in the four groups of cows. Time 0 indicates the time of PGFza administration Group A (n = 4) 11.12 11.6+ 13.4% 10.5+ 16.9 + 20.3 + 31.8+ 23.5 + 41.0+ 44.8 + 67.9 + 48.3+
0.9 0.7 0.8 1.5 2.9 2.4 8.5 2.9 9.5 13.9 22.4 6.3
ab zb ab cd cd cd ab
44.7+ 7.0 16.1 + 1.6 10.9+ 14.32 1.7 1.6 cd 11.3+ 2.4 ab 15.8 & 2.7
Group B (n = 4)
Group C (n = 4)
7.3 + 1.4 9.3 + 0.2 11.9k2.4 8.9+ 1.4 11.25 1.5 10.3 + 1.9 13.1 + 3.6 13.4k2.0 15.9 + 2.8 23.Ok4.2 25.7 + 7.2 25.854.7 28.2 + 8.1 15.8 + 1.2 11.4 + 0.8 13.7 + 2.7 12.2 + 4.2 14.8 + 6.7
8.4+ 3.0 lO.O+ 0.2 14.7 + 5.2 16.8 * 3.5 14.2+ 2.0 16.2+ 2.3 37.2 + 11.4 47.5 * 15.3 69.0+ 15.8 76.7 + 13.7 106.02 7.8 81.0226.9 63.9 + 28.0 53.3 + 41.9 29.5 + 20.5 7.5+ 1.9 4.5 + 1.2 5.2+ 0.1
a z c” ; a cd ab
Group D (n =3)
b ab b b d d d b
c a
9.6+ 1.6 12.9 + 0.6 13.3 + 1.8 11.1+0.9 11.5kO.8 17.7 22.7 14.220.6 15.3 + 2.7 18.0 + 2.4 17.7k2.0 16.2 + 3.3 13.3 + 1.5
a E ; a d b
El Means with different superscripts on the same coloumn statistically differ at P
JUNE 1991 VOL. 35 NO. 6
THERIOGENOLOGY
120.
105.
Group A
90.
(3000 IU PMSG)
75. 60. 45. 30. 15.,yfffy-y 0 -46 -32 -16 120 1 105.
0
16
32
40
64
60
Group B
90.
=
E
-
60.
g
45.
;
:;j&r
E
12OJ -46
2
105. 90.
:
75.
L b-
(loo0 IU PMSG + hMG: 297 NJLH and 297 IU FSH given in SCVC~ decreasing dosages)
75.
-32
-16
0
16
32
46
64
60
Group C (hMG:1575 IU LHand 1575 IUFSH given in nine decreasing dosages)
60. 45. 30. 15. 0 -46 120 1 105.
-32
-16
0
16
32
46
64
60
Group D
so.
(Control)
75. 60.
-46
-32
-16
0
16
32
46
64
60
Hours from PGF,Admlnistration
Figure 1. Variations in the blood concentrations (if SEM) of total nonconjugated estrogens in the four groups of cows. Time 0 indicates the time of PGFzcl administration.
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1163
THERIOGENOLOGY
20. 18 _ 16.
Group A (3000IU PMSG)
14 _ 12. 10. 8. 6. 4. 2. 0 -46 202
-32
-16
0
32
46
64
80
18. 16.
Group B (1000IU PMSG
+ hMG: 297 IU LH and297 IUFSHgiven in seven decmsing dosages)
14. 12 _ IO.
\
8. 6. rE
4. 2.
E
O~~.,~..,........., -48 -32 -16 0 20,
* A
18.
16
32
48
64
80
16 -
Group C
14.
(hMGA575 IU LH and 1575 IU FSH given in nine decreasing dosages)
12. 10 8. 6. 4.
-43
-32
-16
0
16
32
48
64
20.1 18 16 i
Group D
14 12. IO _ 8. 6. 4. 2. -48
-32
-16
0
16
32
48
64
80
Hours from PGF,Admlnlstralion
Figure 2.
1184
Variations in the blood concentrations (Z f SEM) of LH in the four groups cows. Time 0 indicates the time of PGF, admiiistration.
Of
JUNE 1991 VOL. 35 NO. 6
the average P4 concentration remained at 1.4M.9 @ml. The average P4 concentration remained below 1 ng/ml until the end of the experimental period (88 h) in all four groups, even though three cases of statistically significant differences were recorded 32, 56 (P < 0.05) and 88 h (P < 0.01) after the PGF, injection, in which Group C had the highest levels. LH The average LH basal concentrations were found to be generally between 0.7 f 0.2 and 1.1 f 2.0 ng/ml in Group A, 0.62 f 0.1 and 1.1 f 0.2 n&ml in Group B; 0.6 f 0.2 and 1.4 f 0.5 @/ml in Group C; and 0.2 f 0.1 and 0.9 f 0.1 in Group D (Figure 2). A significant difference (PcO.01) between the LH basal concentrations of Group A (0.9 f 0.1 @ml) and Group D (0.3 f 0.1 ng/ml) was seen only 88 h after PGF,, adminstration.The LH preovulatory peaks showed average maximum values 48 h (Group A), 40 h (Group B), values injection, with average 32 h (Group C) and 72 h (Group D) after the PGF of 8.9 f 4.3, 11.6 f 6.3, 13.3 f 12.2, and 7.4 k’P.8 @ml, respectively. The results in Table 2 show the effects of the superovulation treatments on the appearance of the estrogens peak and LH preovulatory peak, and on AI times. In the three treated groups, the estrogens and LH peaks and time of AI were significantly advanced (P&01, PcO.01 and kO.05, respectively) with respect to the control group. The LH peaks occurred after the estrogens peaks at intervals of 2 to 8 h, and they were of reduced amplitude due to the low frequency of blood sampling. Table 2.
Time intervals (hours) between PGF administration estrogens and LH peak and the first AT
Groups A B D C
Estrogens (hours) 38.0 + 3.8 a 42.7 + 5.3 a 69.3 34.0 + 5.3 3.8 ;
(l!Zrs) 46.0 + 5.0 a 44.0+ 2.3 a 41.0+5.2 72.0 + 4.6 6
and the appearance of
First AI (hours) 63.0 + 3.0 ’ 60.0 + 4.9 c 54.0 80.0 + 4.0 3.5 ;
: i Means with different superscripts on the same coloumn statistically differ at P < 0.01. Means with different superscripts on the same coloumn statistically differ at P < 0.05. Group A was given 3000 IU PMSG; Group B received 1000 IU PMSG + hMG (297 IU ofLH and 297 IU of FSH given in seven decreasing doses); Group C received hMG (1575 IU of IJ-I and 1575 IU of FSH given in nine decreasing doses); Group D was the control. Ova Collection Ova were collected from the superovulated cows and were evaluated for embryo transfer suitability and classified as morule, blastocysts, degenerate ova or nonfertilized ova. The average number of ova recovered from Group B was significantly different (P&.01) from that of Groups A and C (Table 3). However, Groups A and C had more nontransferable ova than Group B (PcO.05). The ratio between transferable and recovered ova (Table 4), expressed by angular transformation, indicates that the Group B treatment, in which 86.2% of the ova obtained were transferable, was the most efficient (PcO.05).
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THERIOGENOLOGY Table 3. Average number @ SEM) of ova recovered from the three treated groups and their morphological classification Ova classification
Ova recovered
Group A (n = 17) x+SEM 11.6+ 1.4 a
Group B (n = 12) x+SEM 5.5+0.5
Group C (n = 24) x+SEM b
11.9+ 1.3 a
Ova transferable morule blastocytes
5.42 1.1 2.9 + 0.7 2.5 + 0.9
4.7 f. 0.8 1.4kO.4 3.3 + 0.8
7.7 + 1.1 3.1 +0.8 4.6+ 1.0
Ova not trasferable degenerated not fertilized
6.2* 1.0 ; 2.8 +0.7 d 3.4 f 0.9
0.8kO.2 ,” 0.4 + 0.2 0.420.7 c
4.2 + 0.9 * 2 oTo.5 cd 2:2zo.7 cd
t i Means with different superscripts in the same row statistically differ at P < 0.01. Means with different superscripts in the same row statistically differ at P < 0.05. Group A was given 3000 IU PMSG; Group B received 1000 IU PMSG + hMG (297 IU of LH and 297 N of FSH given in seven decreasing doses); Group C was given hMG ( 1575 N of LH and 1575 IU of FSH given in nine decreasing doses); Group D served as control. Table 4. Absolute number and percentage of ova recovered from the three treatment group. Ova classification
Group A (n =17) n %
Group B (n =12) n %
Group C (n =24) % n
Ova recovered
198 100.0
65 100.0
285 100.0
Ova transferable morule blastocystes Ova not trasferable degenerated not fertilized
93 49 44
47.0 a 24.7 22.3 a
56 16 40
86.2 b 24.6 61.6 b
185 75 110
64.9 b 26.3 38.6 ab
105 47 58
53.0 ; 23.7 29.3 b
9 4 5
13.8 a 6.2 a 7.6 a
loo 48 52
35.1 b 16.9 ab 18.2 ab
a b Means with different superscripts in the same row statistically differ at P < 0.05. Group A was given 3000 N PMSG; Group B received 1000 N PMSG + hMG (297 IU of LH and 297 N of FSH given in seven decreasing doses); Group C was given hMG (1575 IU of LH and 1575 N of FSH given in nine decreasing doses); Group D served as control.
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THERIOGENOLOGY DISCUSSION The analysis of the results demonstrates clearly the intense ovarian stimulation induced by the superovulation treatments. The blood concentrations of the estrogens appeared to depend on the dosage of gonadotrophin used, and in the treated groups these steroids had higher average concentrations than in the control group, with highly significant differences for Group C. Blood estrogen concentrations showed the largest variations within Groups A and C. The estrogen concentrations measured in Group C treated with hMG were similar those reported by McGowan et al. (32) but different from that of other authors (18,33) who evaluated the steroid formation by measuring the concentrations of estradiol-17j3. However, it seems that estradiol-17P shows decidedly lower variations with respect to estradiol-17a and estrone; thus, the simultaneous measurement of estradiol- 1701 and estradiol-17 p is a better method for the evaluation of ovarian stimulation by exogenous gonadotrophins (34). Another effect of the intense ovarian stimulation due to exogenous gonadotrophins could be observed in the persistence of slightly higher P4 levels after treatment with PG in Group C, which in some cases were statistically significant. In this phase, the persistence could be due to the formation of atypical luteinous structures in the ovary. The P4 levels recorded in the later phases of the experiment could be associated with a return to function of the corpora lutea. The hormonal peaks in Group B animals occurred within a narrow time interval; perhaps due to a higher degree of the synchronization of ovulation. Basal levels of LH did not show a significant differences within groups except than that observed 88 h after PG treatment between Group A and Group D. This observation cannot easily explained. The analysis of the data referring to the production of ova in the three groups shows some interesting differences. The PMSG + hMG treatment, in which a less intense steroidogenesis was observed, was characterized by a lower ova production and lower variation. The evaluation of the percentage of transferable ova reveals a higher efficiency of superovulation in Group B. As already stated, the higher degree of synchronization in Group B animals would appear to be further confirmed by the prevalence of one of the two phases of embryonic development classified. With reference to the total number of ova recovered, our results are similar to those of other authors (35, 36), given the doses of gonadotrophin used are taken into account. The number of degenerated ova was decidedly lower in Group B than in Groups A and C. This could have been the consequence of the hormonal and physiological status as shown by the P4/esttogen ratio. Furthermore the hypotesis that the hormonal variations due to the superovulatory treatment may adversely affect sper and/or ova transport, thus interfering with fertilization, has to be considered. It was not possible to establish any correlations between treatments and the percentage of infertile ova because of the practical problem of not being able to use the same semen for AI. The treatment with PMSG or with hMG both provoked a large increase of estrogen concentration, although this response was better controlled in the case of hMG. Clinical observations of PMSG treatment revealed increases in ovary volume to the size of an orange, and cows treated with PMSG showed more irregularities in returning to normal cyclic activity, with a number of cases of ovarian cysts (25). For these reasons, PMSG treatment should not be used in cows which are expected to remain in the herd for some time. From an analysis of our results, and considering the low cost of the PMSGRMG treatment (Group B), we recommend it as an alternative to the very expensive treatment using hMG alone.
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REFERENCES 1. Betteridge, K.J. and Smith, C. Extending the use of embryo transfer in farm animals. Proc. 1 lth Int. Cong. on Animal Reproduction and AI(Dublin), Y: 255-264 (1988). 2. Pallares, A., Zavos, P.M. and Hemken, R.W . Fertilization rates and embryonic development in superovulated cattle inseminated in different sites within the reproductive tract. Theriogenology %:709-719 (1986). 3. Bevers , M.M. and Dieleman, S.J. Superovulations of cow with PMSG: variation in plasma concentration of progesterone, oestradiol, LH, cortisol, prolactin and PMSG and in number of preovulatory follicles. Anim. Reprod. Sci. fi:37-52 (1987). 4. Callesen, H., Greve, T. and Hyttel, P. Preovulatory endocrinology and oocyte maturation in superovulated cattle. Theriogenology 25:7 l-86 (1986). 5. Chupin, D., Procureur, R. and Combarnous, Y. Effect of LH on FSH induced superovulation in four breeds of cows. Proc. 3rd Meeting A.E.T.E., (Lyon) : 15 1 abstr. (1987). 6. Oliva, O., Cremonesi, F., Crotti, S. and Lauria, A. Incidenza della razza, dell’ed e de1 tipo di trattamento sulla superovulazione della bovina. Atti Congresso SlSVet, XXXVI: 310-313 (1982). 7. Armstrong, D. T. and Evans, G. Factors influencing success of embryo transfer in sheep and goats. Theriogenology B:3 l-42 ( 1983). 8. Armstrong, D. T., Pfitzner, A. P., Warnes, G. M., Ralph, M. M. and Seamark, R. F. Endocrine responses of goats after induction of superovulation with PMSG and FSH. J. Reprod. Fertil. 41: 395-401 (1983). 9. Nuti, L. C., Minhas, B. S., Baker, W. C., Capehart, J. S. and Marrack, P. Superovulation and recovery of zygotes from nubian and alpine dairy goats. Theriogenology 28: 481-488 (1987). 10. Lemer,S.P.,Thayne, V.W.,Baker, R.D.,Henschen, T.,Meredith, S., Inskeep, E.K., Dailey, R.A., Lewis, E.P. and Butcher, R.L. Age, dose of FSH and other factors affecting superovulation in Holstein cows. J. Anim. Sci. &&176-183 (1986). 11. Moor, R.M., Kruipp, Th.A.M. and Green, D. Intraovarian control of folliculogenesis: limits to superovulation ? Theriogenology a: 103- 116 (1984). 12. Christie, J.B., Newcombe, R. and Rowson, L.E.A. Ovulation rate and egg recovery in cattle treated repeatedly with pregnant mare serum gonadotrophin and prostaglandin. Vet. Rec. @&281-283 (1979). 13. Donaldson, L.E. and Perry, B. Embryo production by repeated superovulation commercial donor cows. Theriogenology 20: 163-168 (1983).
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14. Saumande, J. and Chupin, D. Induction of superovulationin cyclic heifers: the inhibitory effect of a large doses of PMSG. Theriogenology x:233-247 (1986).
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