Ovarian response to superovulation in Nelore cows (Bos taurus indicus L.)

THERIOGENOLOGY

OVARIAN RESPONSE TO SUPEROWLATION IN NELORE COWS (Bos taurus indicus L.) --W.A.P. Becker' and L.E.L. Pinheiro

2

1 Agropecu&ia Lagoa da Serra 14160-Sertzozinho-SP-Brazil 2

Department of Clinic and Surgery, FCAV-UNESP, 14870-Jaboticabal-SP-Brazil Received fur publication: October1, 1984 Accepted: April

25, 19%

ABSTRACT Superovulation was induced in 15 Nelore cows with porcine follicle stimulating hormone (FSH-P) or pregnant mare serum gonadotropin (PMSG), and embryos were collected to compare the response of this breed of cattle to these hormones. FSH-P was given in 30-and 40-mg doses for 5 d as a single daily injection or fractionated into two daily injections. PMSG was given in doses of 1000 and 2000 IU. The animals were killed immediately after embryo collection and the ovaries and genitalia were examined clinically. PMSG proved to be more effectivein inducingsuperovulation than FSH-P, probably because PMSG caused no stress since it was administered as a single dose. No differences were observed between the 30-and 40-mg dose of FSH-P or between the application as a single or fractionateddose. Differences did occur, however, between the number of ovulations and embryos obtained at each collection. On the basis of postmortem analysis, we concluded that lack of egg uptake by the infundibulum had occurred in cases of increased ovulation, with excessive increase in volume of the ovary. We EilSO recommend using smaller doses of FSH-P and suggest that avoiding stress in handling is essential for a good response to hormonal stimulation by Zebu cattle. Key words: Nelore,

superovulation,

FSH-P, PMSG

INTRODUCTION As embryo transfer becomes increasingly important,there is a greater need for basic information on reproductive Acknowledgments The authors are grateful to CNPq/FAPESP-Brasil for financial support and also wish to thank Dr. C. Gropp SP, Brazil. Sertzozinho, Agropecusria Lagoa da Serra,

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THERIOGENOLOGY physiology. When superovulation is induced, for example, the doses and hormones used are based on a somewhat empirical scheme, especially among Zebu cattle raised in the tropics. Elsden and Kessler (l), working with 24,Nelore cows in a tropical region, demonstrated that 24 mg FSH administered in fractionateddoses for 4 d (4,3,3 and 2 mg FSH twice daily) was more effective than 50 or 36 mg applied in the same manner. Extensive information has been published on the types and doses of gonadotrophins used for superovulation. FSH or PMSG is commonly used. When FSH is used,repeated injections are needed because of the short half-life of the hormone (about 110 min; 2, 3). In contrast, a single application of PMSG induces satisfactory‘responses. According to Schams et al. (41, PMSG has a long half-life that is additionally affected by the molecule components. There is a component with a shorter half-life (40 to 50 h) and one with a longer half-life (118 to 123 h). Furthermore, the FSH-IH (luteinizing hormone) effect ratio in PMSG is 1.08, i.e., a high LH concentration that thus leads to early follicle activation (5, 6). Another important factor is the variability of gonadotrophic preparations. When evaluating the usual sources such as equine chorionic gonadotrophin, porcine FSH, and human menopausal gonadotrophins, Murphy et al. (7) detected obvious variations in the FSH-LH ratio between preparations and between lots within a given preparation. In turn, Chupin et al. (8) demonstrated that prepasations with lower LH fractions induce better ovarian responses, although these responses vary widely among different breeds (9). In Zebu cattle, some variables such as duration of estrus and interval from estrus to ovulation are still unknown. In view of these considerations, the present study was carried out on Nelore cows to determine the ovarian responses to stimulation with different doses of FSH and PMSG. The efficiency of the collection method and the level of accuracy in detecting the structures on the ovaries by rectal palpation were also evaluated. MATERIALS AND METHODS The animals used were 15 clinically healthy, uniparous Nelore cows, specially selected for an embryo transfer program. Each animal, with a palpable corpus luteum (CL) on the ovary, received two doses of cloprostenol (500 ug) 11 d apart. Immediately after synchronization, superovulation was induced with FSH-P and PMSG. Starting on Day 9 of the cycle (estrus= day O),two donors were injected with 3 mg FSH twice daily for 5 d and four donors were injected with 6 mg FSH once daily for 5 d. In a second group of animals, 8 mg

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THERIOGENOLOGY

PSH was injected daily with two donors receiving a single injection daily. Total dosage for each group was 30 and 40 mg, respectively. A third group consisted of animalsreceiving 1000 IU (1 animal) and 2000 IU (2 animals) of PMSG in a single intramuscular injection on Day 9 of the cycle. Cloprostenol was applied again on Day 11 of the cycle. All animals showed signs ofestxus 2 d after the application of the prostaglandin F2o (PGF2a) analogue and were inseminated 0, 12, and 24 h after estrus was detected. Embryos were collected 7 d after the last insemination by a nonsurgical technique using a Neustadt/Aisch catheter. The uteri were flushed with phosphate buffered saline (PBS) containing 1% fetal calf serum (FCS). The embryos were allowed to settle for 15 min at 37OC before the supernatant fluid was siphoned. Using an Olympus stereomicroscope, the oocytes and embryos were classified according to their appearance as unfertilized, fertilized normal and fertilized abnormal. Immediately after collection, three technicians with extensive clinical experience rectally palpated the animals and estimated the number of CL and follicles present in each ovary. These data were recorded fox c~ariscn withpostmortem results. When the animals were slaughtesed, their reproductive tracts were collected and the ovaries were examined for CL and follicles. The ovarian structures were counted, classified,and photographed. The study consisted of a 2 by 5 factorial design tested by Chi-square. The factors were as follows: Treatment 1, 30 mg FSH-P (single daily dose of 6 mg); Treatment 2, 30 mg FSH-P (3 mg twice daily); Treatment 3, 40 mg FSH-P (single daily dose of 8 mg); Treatment 4, 40 mg FSH-P (4 mg twice daily); and Treatment 5, 2000 IU PMSG (single injection). The responses considered were viable and degenerated embryos. RESULTS In only 12 animals that received FSH, 247 ovulations occurred,However, only 86 embryos (20 viable and 66 degenerated) were collected, representing 2.9 ovulations per embryo. Of the animals that received 2000 IU of PMSG, 25 ovulations occurred and 19 embryos (15 viable and 4 degenerated) were collected, representing 3.4 ovulations per embryo. The recovery rate was higher in the PMSG group (76%) than in the FSH group (35%), although the ovarian response was lower with PMSG. The total values obtained after application of the FSH-P doses administered daily as a single or divided dose and after application of PMSG are presented (Table 1). These values were arrangedto determinestatisticaldifferences among

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THERIOGENOLOGY

treatments (Table Z).Data for the animal that receivedonly 1000 IU of PMSG are not presented since they were negative. Table 1, Ovarian reaction and embsyo quality after administration of different doses of FSH-P in single or divided doses and after a single injectionof PMSG Treatment

structwres

FSH

PMSG

!&ice daily

Number of donor CL

Cr~cedaily

Cne injecticn

3mg

4mg

6mg

8mg

2000 IU

2

2

4

4

2

17a

27.5

20

19.5

12.5

Follicles

1.5

Viable embryos

OWb 3.5

3.5(27) 1.0(14) 3.0(55) 9.5

6.25

2.5

2.0

3.5

13.0

7.25

5.5

9.5

Deg.esnbryos !I.WaleKbrycs Percent recovery

3.0

21

47

5.5

36

2.75

3.5

28

7.5(79)

76

aAveragenumber per donor. bNurker in parenthesesare percentages. Table 2. Values obtained using a 2 by 5 factorial design chisquare analysis. The number of viable and degenerated embryos was considered for each treatment. Canparisan between treatments

Level of

Chi'

signifiCanCe

-

Canpariscn betsEen treatments _

Chi'

Level of signifiCanCe

XZa l-3

1.1960 0.5323

NS NS

2-4 2-5

1.2939 5.9453

NS *

l-4 l-5

2.8636 6.5311

NS *

3-4 3-5

2.4124 6.9838

NS *

2-3

0.4642

NS

4-5

0.9877

NS

aTreatment Treatment Treatment Treatment Treatment *I? -z 0.05.

788

1: 2: 3: 4: 5:

30 mg FSH-P 30 mg FSH-P 40 mg FSH-P 40 mg FSH-P 2000 IU PMSG

(single daily dose of 6 mg). (3 mg twice daily). (single daily dose of 8 mg). (4 mg twice daily). (single injection).

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THERIOGENOLOGY

A comparison of the responses of the left and right ovary after various treatments showed that the right ovary responded most effectively (Table 3). The mean values of the clinical examination were obtained by computing the three evaluations made simultaneously by the three technicians. Fox technical reasons, this comparison was made for only 13 animais. Only follicles larger than 5 mm in diameter were included in the calculations at the time of inspection. Table 3. Comparison of the number of corpora lutea (CL) and follicles (F) detected by clinical evaluation and by postmortem examination PostmortemInspection -

Animal No.

Left

1

4CL/lF

XL/=

3

5WJF 4CL/2F

8a/lF

4 5 6 7 8 9

9CL 5CL 7CL/lF 9a 6CL/lF

Il.

7CL

13

5CT.J

14

9C.G

15

5CL

16

8a

l&in

Right

=/LF

5CL/2F

9a

llCL/3F

~CL/~F

5CL/lF

llCL/4F

13CL

28CL/2F

5CL

4cL/1F 5CL/5F

5CL/2F

9CL/2F 8CL/2F

17CL/lF

5cL

7CL 7CL/3F 4cL/LF 16CL/2F 16CL 6CL

6.4CL/O.5F 8.1CL/1.2F

7CL/3F 15CL/2F

8CL/2F

lla/D

8CL/7F 4CL/lF

13CL/4F

15CL/2F

29CL

12a 8CL/2F

9CL/3F lla 7u/2F

8.8CL/l.8F 12.2CL/1.9F

The difference in thenunbers obtained varied between the two methods used for the assessment of ovarian response. For example, 27% more CL and 72% more follicles were observed on the left ovary at postmortem. Similarly, 33% and 37% more CL and follicles, respectively, were observed on the right ovary. All ovaries were cut lengthwise for the assessment of the CL and follicle walls. Eight point one

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THERIOGENOLOGY

percent of the CL exhibited an internal cavity, 10% of which were classified as lutcal cysts; 11.9% of the follicles, exhibiting luteinization, were identified as luteinized follicles. These structures were visualized only in FSHtreated ovaries (Figure 1).

Figure 1. Two ovaries showing the effects of FSH-P stimulation. A, shows a good response (more than 12 CL, including some unruptured follicles). B shows cystic CL.

When the reproductive tracts were examined, all uterine horns showed hemorrhagic sites in the subserosa. Hemorrhagic areas were also noted in the mucosa and submucosa and coincided with the site of‘location of the catheter balloon.

790

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THERIOGENOLOGY

DISCUSSION The differences in ovarian response with respect to the number of CL and the embryos collected can be explained in various ways: 1) lack of egg uptake by the infundibulum due to the intense ovarian reaction, 2) premature or delayed ovulation, 3) delayed egg or embryo descent because of imbalance in the gonadal steroids, 4) accelerated descent into the uterus because of this same ovarian hormone imbalance. All genitalia were flushed immediately after slaughtering, and no remaining embryonic structures were detected. Thus, the most plausible explanation may be the first, i.e., lack of egg uptake by the infundibulum. The cause of the large proportion of degenerated embryos may have been the imbalance of stesoids produced by the CL and by the follicles. Figure 1 shows some CL in different stages of development, meaning that ovulation was not synchronized. There were two characteristic cysts of the CL and unruptured luteinized follicles (frequency 10% and 11.94, respectively), reinforcing the possibilityof a hormonal imbalance and explaining why no large number of partially degenerated embryos was found when PMSG was applied, since the number of ovulations and altered structures is smaller with the use of PMSG. Booth et al. (10) believe that the uterine environment becomes unreceptive t&conception when the steroid levels are changed. Comparison between the various doses and between forms of application (once or twice daily), was impaired by the small number of animals. Even so, we obsesve from the data that it does not seem to matter whether application is made in fractionated doses or not. After the fractionated doses, 33 embryos (out of 89 ovulations)were collected; i.e., 2.7 ovulations were needed to obtain one embsyo. When the same doses were applied singly, 51 embryos [cutof 158 ovulations) were collected; i.e., 3.1 ovulations were needed to obtain one embryo. The Chi-square analysis showed that there are not statistical differences between dosages (30 or 40 mg) and between fractionated or singleinjection. However, the cows were nervous, and every time they were restrained to receive the hormone, intense stress was triggered. Thus, it is possible that by injecting only a single daily dose, a response equivalent to the injection of two fractionated doses was obtained simply because the negative effects of stress were decreased. This is not the case for Taurine cows, as pointed out by Monniaux et al. (3). The lack of stress may also have contributed to the SUCCESS obtained with a single application of PMSG; this possibility deserves further study. Although the statistical test showed a difference (P < 0.05) between FSH and PMSG treatments, the group receiving PMSG had only two animsls,which was a severe limitation. As expected, the right ovary responded to treatment more intensely than the left one, no matter what treatment was applied. JUNE 1986 VOL. 25 NO. 6

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THERIOGENOLOGY

In most cases, ovarian responses are assured by rectal palpation, This technique is subject to error (Table 3), since many structures do not exhibit prominences on the external surface of the ovaries or even simulate a superimposition. The three technicians that performed the clinical evaluation were highly experienced and performed their examinations separately to avoid subjective errors.The estimated error for the left ovary (27%) was smaller than that for the right ovary (33%), since the reaction of the last one was much more intense. Another factor worth mentioning is that when the ovaries showed a smaflerreacticn (seven or eight structures), the error was also smaller. Above 10 structures, the error tended to be grosser. The uterine lesions were a constant finding and were probably caused by thecatheter. Since technician experience was well known, the possibility of untimely maneuvers was to maintain maximum asepsis ruled out, This shows the need and to take special care in handling the uterus, especially In nervous animals. Many of the data obtained in the present study need further confirmation. However, in terms of Braziliantropical conditions, the data obtained by Elsden and Kessler (1) and in the present study summarize the experience on superovulation induction in Zebu cattle in the Brazilian tropics and allow us to state that superovulation schemes must be carried out along special lines, with practical and structural differences from those used for Taurines. REFERENCES 1.

Elsden, R.P. and Kessler, R.M. Superovulation of Nelore cows and heifers. Theriogenology 19:127 abstr. (1983).

2.

Patterns Gay, V-L., Midgley, A.R. and Niswender, G.D. of gonadotrophic secretion associated with ovulation. Fed. Proc., Fed. Amer. Sot. Exp. Biol. -29:1880-1887 (1970).

3.

Superovulatory Monniaux, D., Chupin, D. and Sahmande,,J. Theriogenology -19:55-81 (1983). responses of cattle.

4.

Schams, D., Menzer, C., Schallenberger, B., Hoffman,B., Hahn, J. and Hahn, R. Some studies of pregnant mare serum gonadotrophin (PMSG) and on endocrine response after application for superovulation in cattle. 12: Sreenin, J.R. (ea.).Control of Reproduction in the Cow. M. Nijhoff, the Hague, 1978, pp. 122-143.

5.

Stewart, F., Allen, W.R. and Moor, R.M. Pregnant mare serum gonadotrophin: ratio of follicle stimulating hormone and luteinizing hormone activities measured by (1976). radio-receptor assay. J. Endocr. 2:317-382

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6.

Papkoff, 1-I. Relationship of PMSG to the pituitary gonadotrophins. &: Sreenin, J.R. (.ed.). Control of Reproduction in the Cow. M. Nijhoff, the Hague, 1978, PP. 122-143.

7.

Murphy, B.D., Mapletoft, R.J., Manns, J. and Humphrey, W.D. Variability in gonadotrophin preparations as a factor in the superovulatory response. Theriogenology -21:117-125 (1984).

8.

Chupin, D., Combarnous, Y. and Procureur, R. Antagonistic effect of LH on FSH-induced superovulation in cattle. Theriogenology 21:229 abstr. (1984).

9.

Chupin, D., Combarnous, Y. and Procureur, R. Different effect of LH on FSH-induced sup&ovulation in two breeds of cattle. Theriogenology -23:184 abstr. (1985).

10.

Booth, W.D., Newcomb, R., Strange, H., Rowson, L.E.A. and Sacher, H.B. Plasma oestrogen and progesterone in relation to superovulation and egg recovery in cows. Vet. Rec. 97:366 (1975).

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