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The production of ova by NewZealand romney ewes followinghormonal stimulationI. A. Cumming a b & M. F. McDonald ba Sheep Husbandry Department, Massey University ,Palmerston Northb S. S. Cameron Animal Research Laboratory ,Werribee, Victoria{cyAustralia , AustraliaPublished online: 12 Jan 2012.

To cite this article: I. A. Cumming & M. F. McDonald (1967) The production of ova byNew Zealand romney ewes following hormonal stimulation, New Zealand Journal ofAgricultural Research, 10:2, 226-236, DOI: 10.1080/00288233.1967.10425130

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226

THE PRODUCTION OF OVA BY NEW ZEALAND ROMNEY EWES FOLLOWING HORMONAL

STIMULATION

By I. A. CUMMING*t AND M. F. McDONALD*

(Received 21 December 1966)

ABSTRACT

Pregnant mares' serum (PMS, 500-1,500 Lu.) was injected into 149 Romney ewes on days 11, 12, or 13 of the oestrous cycle following induction of ovulation by injections vf progesterone. The ewes were mated to fertile rams and recovery in vivo of ova attempted 55-91 hours after onset of oestrus.

PMS hastened the onset of oestrus and caused super-ovulation. Marked variation in ovarian response existed, especially after injection of 1.500 i.u. PMS.

The percentage of ova recovered ",as not associated with variation in number of corpora lutea, but the fertilisation rate decreased signifi-cantly when more than about 12 ovulations had occurred.

The stages of cleavage of ova relative to interval from onset of oest-rus were recorded. Recovery of ova at about the 8-ceJl stage was most successfulIy attempted 72 hours after omet of oestrus and when the ewes were observed at 8-hourly intervals.

INTRODUCTION

Coincident oestrus and ovulation have been induced in New Zealand Romney ewes by injection of progesterone and/or gonado-trophin during the breeding season (Wallace 1954; Wallace, Lambourne. and Sinclair 1954) and anoestrous season (Lambourne 1955; Raeside and Lamond 1956; Edgar 1958; McDonald 1961). Considerable variation in ovulation response to gonadotrophin (especially at high-dose levels) has been found with various breeds of sheep (e.g. see Averill 1958); thus, with the Romney ewe, Wallace (1954) reported that 1,000 i.u. Pregnant Mares' Serum gonadotrophin (PMS) when injected during the oestrous cycle has caused from one to 13 ovulations per animal. The existence of such variation in ovarian response is a major limitation to the usefulness of hormonal treatment for increasing the fecundity of sheep.

Hormonal injection of ewes to cause the release of many ova is commonly used for experimental purposes. During a study of prenatal mortality in Romney sheep, which involved the use of the * Sheep Husbandry Department, Massey University. PaImerston North. t Present address: S. S. Cameron Animal Research Laboratory, Werribee. Vic-

toria. Australia.

N.Z. Jl agric. Res. (/967), 10: 226-36

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1. A. CUMMING AND M. F. McDoNALD 227

technique of ovum transplantation, the opportunity arose to examine the oestrous and ovarian responses in "donor" ewes following pro-gesterone and PMS injection. The purpose of this paper is to report:-

1. The method used for the production of fertilised ova in the Romney ewe.

2. Factors which influence the response to PMS gonadotrophin.

MATERIALS AND METHODS Animals and their management

One hundred and fifty-four New Zealand Romney ewes, aged four to six years, and drawn from the flocks of Massey University, were used during February-April in either 1964 or 1965.

Ewes were identified by ear tags and numbers branded on their sides. The animals grazed ryegrass-white clover pasture and were managed to maintain constant bodyweight. Liveweights were recorded at weekly (1964) and fortnightly (1965) intervals.

Vasectomised rams and rams of proven fertility and all fitted with Sire Sine harnesses were kept with the ewes as required. The ewes were inspected for mating marks every 12 hours when with "teaser" rams and at eight-hourly intervals when entire rams were present.

Hormones and treatment To attempt to induce and synchronise oestrous cycles of ewes

(prior to experiments involving egg transplantation) 13-15 daily injections of progesterone were given as described in the other paper in this issue (Cumming and DcDonald 1967). Vasectomised rams were with all ewes from a week before treatment until after oestrus or "silent" oestrus had occurred, and entire rams were subsequently joined with the ewes to allow fertile services at the next oestrous period.

To induce super-ovulation at that time, PMS* (500-1,500 i.u.) was subcutaneously injected on days 11, 12, or 13 after oestrus. Where oestrus had not occurred following progesterone "synchronisation" an estimate of the time of "silent" oestrus was made.

Recovery of ova Laparotomies were conducted on 147 ewes 55-91 hours after the

onset of oestrus and mating to entire rams. In each animal the reproductive tract was exposed, and numbers of corpora lutea and large follicles recorded. Ova were recovered by flushing the Fallopian tubes and the upper portion of the adjacent uterine horn with sheep serum using essentially the same procedure as that described by Hunter, Adams, and Rowson (1955). The washings were immediately examined for ova, and details of their number, stages of cleavage, and appearance noted. * PMS - Boots Pure Drug Company.

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228 Hormonal stimulation and ova production

TABLE I. Lengths of oestrous cycles in ewes*, relative to day of PMS treatment

1964 Season r 1965 Season

Day of I f I Cycle N f Cycle injection No. 0 length o. 0 length

ewes I (days) ewes (days)

11 5 15.0 ± 0.2

12 20 15.4 ± 0.2 52 15.7 ± 0.1

13 14 16.1 ± 0.4 3 16.1 ± 0.3

* Data for ewes treated after "silent" oestrus are excluded.

TABLE 2. Ovulation response and percentage of ova recovered, relative to dose of PMS ----,--

Dose No. of iN o. of corpora lutea I Percentage Season PMS ewes of

(i.u) treated ! (Mean ± S.E.) ova recovered

1964 0 2 1.50 100 1000 43 3.58 ± 0.36 79 1200 12 3.08 ± 0.65 84 1500 3 9.00 ± 5.71 74

1965 0 3 2.00 50 500, 900 2 2.00 75

1000 50 5.02 ± 0.50 68 1500 27 8.52 ± 1.06 75

TABLE 3. In vivo recovery of ova from Romney ewes

No. of ewes No. of I corpora lutea I in ewes in Percentage

Season Subjected which ovum of ova Flushed Yielding recovered to for ova recovery was

laparotomy ova attempted

I I i 1964 I

63 60 59 I

217 76

1965 I 84 I

81 79 I 489 71 I I I

I Both Seasons 147 141 138 706 72.5

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I. A. CUMMING AND M. F. McDoNALD 229

RESULTS Length of oestrous cycles

Table 1 shows the mean lengths of oestrous cycles in ewes treated with PMS (all doses combined) relative to the day of injection. There was a significant decrease (p < 0.01) in the length of the cycle associated with advancement in the day of injection. Examination of these data for treatments within days of the cycle showed no evidence that the cycle length varied according to the quantity of PMS administered.

The effect of PMS in shortening the oestrous cycle is also revealed when comparison is made with data for 143 ewes not treated with gonadotrophin for which the cycle length after progesterone "synchronisation" was 16.4 -+- 0.2 days.

Ovarian response In the 1964 and 1965 seasons. one and two ewes respectively were

found with cystic ovaries and each contained several large follicles. Excluding these three sheep. the ovarian responses to PMS in the remainder of the ewes are shown in Table 2. In both years the increase in ovulation rate associated with rise in quantity of PMS injected (i.e. 1,000 i.u. to 1.500 i.u.) was statistically significant (p < 0.01). The ovarian response appeared independent of whether the PMS was administered on the 1 Ith. 12th, or 13th day of the cycle. Likewise, whether oestrous behaviour occurred or did not occur - following progesterone treatment had no effect on the ovulation rate after the PMS injection.

Recovery of ova Laparotomies were conducted on 147 ewes to attempt to recover

ova (Table 3). In addition to the three animals with cystic ovaries, another three showed abnormalities in the genital tract; these were distended Fallopian tubes. blockage at the utero-tubal junction. and a ewe in which the ovaries could not be exposed to the exterior. A further ewe was examined when the appearance of the corpus lutem indicated ovulation had occurred about 8 days prior to laparotomy; ovum recovery was not attempted.

Fig. I shows the percentages of ova recovered relative to the number of corpora lutea, which is in turn a reflection of the dose of PMS administered. The data show that at least up to approximately 20 corpora lutea or 1,500 i.u. PMS no significant effect on the number of ova recovered in vivo was detected.

Operations to attempt the recovery of ova were conducted on ewes from 55 to 91 hours after the onset of oestrus. These times were chosen so that ova would be collected from within the Fallopian tubes and preferably at the 8-cell stage of cleavage or even at more advanced stages. Table 4 shows that there was no decrease in the percentages of ova recovered as the interval from the onset of oestrus to laparotomy increased.

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230

100

Q w 80 I cJ)

~ 0 60

LL 0 I-

40 z w u 20 a:: w a..

Hormonal stimulation and ova production

OVA

~: UNCLEAVED ...., LOST - i

1964

1 2 3 4 5

No. OF CORPORA LUTEA

CLEAVED .... 1965

6 7-11 12-23

PER EWE

Fig. I.-Percentages of ova recovered and fertilisation rate, relative to number of ova shed.

TABLE 4. Recovery of ova relative to the interval to laparotomy after onset of oestrtf,

Interval to recovery of ova after , onset of oestrus (hours)

Se:lson \ 55-60(61-661\67-72173-78179-84 :-85---90-1-9-1-96

1 I : ----, -----~r- ,--------------1 1964 I Number of ewes I 12 17 13 17 I

I Percentage ova ' , I recovered 73.1 72.6 79.7 72.6 83.3

1965 'Number of ewes 3 9 13 29 22 5 I

Percentage ova I I recovered 1 70.4 88.3 78.8 58.3 I 74.4 70.6 100.0

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I. A. CUMMING AND M. F. McDoNALD 231

Fertilisation of ova Of the 506 ova recovered at laparotomy, 66% were fertilised.

A significantly greater proportion of cleaved ova was obtained in 1964 (81%) than in 1965 (58%) (X 2 = 26.3; P < 0.001).

Fig. 1 shows the percentages of fertilised ova relative to the number of corpora lutea. These data suggest that at least up to about 10-12 ovulations after PMS treatment the fertilisation rate was satisfactory: a decrease in proportion of cleaved ova was shown in ewes which had between 12 and 23 corpora lutea. The effect of a large number of corpora lutea on fertilisation rate was almost one of either all or no ova undergoing cleavage. For example, in the two seasons 18 sheep responded to PMS with more than 10 corpora lutea per ewe; and of these animals one ewe gave both cleaved and uncleaved ova at laparotomy, 7 ewes gave only cleaved ova, and 10 ewes gave only uncleaved ova.

Table 5 shows the numbers of ewes from which cleaved or uncleaved ova, or both types, were recovered relative to the dose of PMS. As a greater percentage of ewes in 1965 were treated with 1,500 i.u. PMS than in the previous year, the increase in ovulation rate was accompanied by a decrease in fertilisation rate. Since the proportions of sheep yielding both types of ova did not vary with dose-level of PMS, the reduced fertility in 1965 was due to an increased proportion of sheep in which all ova were uncleaved. The difference in proportion of ewes which produced solely uncleaved ova after 1,500 i.u. PMS and after 1,000 i.u. PMS respectively was statistically significant (X2 = 11.2; p < 0.01). During the two seasons there were 16 sheep with both cleaved and uncleaved ova and in 9 of these animals both types of ova were recovered from within the

TABLE 5. Numbers of ewes yielding cleaved, uncleaved, or both cleaved and uncleaved ova relative to the dose ot PMS

Number and percentages* of ewes yielding PMS Number

Season dose of ewes I I Both cleaved (i.u) examined N ,Cleaved ova Uncleaved and un cleaved I 0 ova i only I ova only ova I

1964 0 2 i 1 1 1,000 43 (2.3%) 33 (76.7%) 6 (14.0%) 3 (7.0%) 1,200 12 8 (66.7%) 3 (25.0%) 1 (8.3%) 1,500 3 3 (100.0%)

1965 0 3 11 I 2 500 1 ill 900 1

1,000 50 12 (4.0%)1 35 (70.0%) 5 (10.0%) 8 (16.0%) 1,500 27 I 11 (40.7%) 12 (44.4%) 4 (14.8%)

I -

* Percentages are of total ewes within a dose-level group within a season.

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232 Hormonal stimulation and ova production

same Fallopian tube. Microscopic examination of the uncleaved ova from ewes in which some eggs were fertilised showed that sperma-tozoa were attached to the zona pellucida of ova in about half of these ewes in each year.

Observations made on the appearance of unc1eaved ova revealed that in two sheep the zona pellucid a of the ova was distinctly involuted. These ova were classed as abnormal. but their appearance might have arisen following degenerative changes owing to failure of fertilisation. All other uncleaved ova were considered to be normal. In most ewes the appearance of the corpora lutea indicated that ovulation had occurred about 3 days prior to laparotomy, yet within some animals on the high dose-level of PMS there seemed to be a proportion of late-occurring ovulations as well as those formed at the normal time. The occurrence of late ovulations could have been an important factor in preventing the fertilisation of the ova, perhaps by affecting transport of spermatozoa to the site of fertilisation. Ova recovered from these ewes should have been at the 4-cell stage and most at a later stage of development. No uncleaved ova recovered from ewes given 1,500 i.u. PMS had any spermatozoa attached to the zona pellucida.

Fig. 2 presents data on the stages of cleavage of ova relative to the interval between onset of oestrus and laparotomy. The earliest times of recovering cleaved ova of each cell stage are also shown.

Altogether 240 eggs which had advanced to more than 6 cells were collected. A significantly greater proportion of ova at these cell stages was obtained from ewes when laparotomies were performed 68 or more hours after oestrus than from ewes examined before this time (x 2 = 3.9; < 0.05). The finding of a number of 2-cell ova relatively late after onset of oestrus was often associated with corpora lutea of 1-2 days ag~ (i.e. younger corpora lutea than might have been expected prior to operation).

DISCUSSION In order that ova for experimental purposes would be available at

specific times. the oestrous periods of donor ewes were controlled by progesterone injections as described elsewhere (Cumming and McDonald 1967). Infertility or reduced fertility following the use of progesterone and PMS has frequently been observed in sheep (reviewed by Lamond 1964). and it has been suggested that progesterone treat-ment is r~;ponsible for the impaired fertility (Davies 1960). To avoid the possibility of lowered fertility the present ewes were allowed one oestrous cycle after progesterone administration. prior to mating and ovum recovery. This procedure has also been adopted by Hancock and Hovell (196 I) for similar reasons. yet this may be unnecessary. as Shelton and Moore (1966) have recently shown that the survival of eggs transferred to recipient ewes was not affected by previous progesterone treatment.

Administration of gonadotrophin during the follicular phase of the cycle hastened the onset of the following oestrus and results agree with data found by other workers. Wallace (1954). while noting an advancement in onset of oestrus following injection of gonadotrophin.

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w (9

~ <t: w ...J U

LL o w (9 <t: ~ (/)

12

8-12

8

6-8

4-6

4

2

I. A. CUMMING AND M. F. McDoNALD 233

e

® 1-1 ---+----1

to>

®

Ii)

•

(i)

60 65 70 75 80 85 90 HOURS AFTER ONSET OF OESTRUS ~ MEAN:': S.D.

\!) EARLIEST RECOVERY

Fig. 2.-Times of recovery of cleaved ova in relation to the onset of oestrus.

found little difference between the cycle lengths of Romney ewes when injected on any of the 10th to 14th days of the oestrous cycle. The present results showed a significant reduction in the cycle length when PMS was given on day 12 rather than on day 13. No relationship existed between the dose of PMS and the cycle length, although such an effect was reported by Wallace, Lambourne, and Sinclair (1954). For effective synchronisation of oestrous cycles in ewes intended for experimental purposes involving ovum transplantation, some considera-tion should therefore be given to the earlier time that oestrus may occur in the donor ewes if they are treated with gonadotrophin to cause super-ovulation. Thus variation in the length of cycle will partially determine the number of donor ewes available to supply ova at any particular time.

The ovarian response to PMS involved a marked increase in the number of multiple ovulations when compared with the normal ovulation rate found in Romney ewes (Averill 1959, 1965; McDonald and Ch'ang

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234 Hormonal stimulation and ova production

]966). In one year, increase in quantity of PMS injected from 1,000 i.u. to ],200 i.u. was accompanied by a decrease in ovulation rate (p < 0.05), yet at the same time 1,500 i.u. given to 3 ewes showed a worthwhile rise in number of ova shed and most were fertilised. It was for this reason that in the second year a greater proportion of ewes were treated at the higher dose-level. Although the number of ova released varied considerably, there was no signficant change in th~

percentage of ova flushed from the genital tract with rise in ovulation rate. In contrast, when the numbers of corpora lutea exceeded approximately 10-12 per ewe, the percentage of fertilised ova recovered declined quite markedly. Thus, the lower fertilisation rate which occurred in the second season was associated with the fact that a higher percentage of ewes were treated with 1,500 i.u. PMS than in the previous year.

Reasons for the failure of fertilisation in some ewes given the high dose of PMS are unknown. Examination of uncleaved ova showed that a few ova might be classified as abnormal, although there was no evidence that such abnormalities were the reason for fertilisation failure. Most of the uncleaved ova appeared to be normal. This result is in agreement with observations of Berry and Savery (1958) and Averill (1958), who concluded that the proportion of abnormal ova from ewes treated with PMS did not exceed the proportion from untreated ewes. It seems likely, therefore, that most ova were viable but were not fertilised owing to insufficient spermatozoa reaching the site for fertilisation. The absence of spermatozoa attached to the zona pellucid a of most uncleaved ova probably suggests interference in the transport of spermatozoa to the Fallopian tubes, but failure due to faulty timing of ovulation and sperm viability might also be implicated. Ewes which received 1,500 i.u. often showed on the same ovary corpora lutea which appeared of variable ages and were usually accompanied by large unruptured follicles.

Laparotomies were conducted to recover the maximum number of ova at approximately the 8-cell stage. Averill and Rawson (1958) recommended that surgery should be performed about 60 hours after the ewe was found to be marked by the fertile ram at morning inspection when the ewes were examined twice daily. Hancock and Hovell (1961) operated a full 72 hours after hand mating and noted that this procedure was effective in avoiding the recovery of ova younger than the 6-cell stage, although this may have increased the percentage which were lost. In the present study, observations for fertile mating were made at 8-hourly intervals, and laparotomy some 72 hours after onset of oestrus showed that most of the ova could be recovered. at

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1. A. CUMMING AND M. F. McDoNALD 235

about the 8-cell stage. However, with high levels of PMS there is often marked variation in times of ovulation, so that 2-cell ova may be recovered together with ova at more advanced stages in development. In addition the low fertilisation rate that often occurs when the ovaries are excessively stimulated may more than outweigh the advantages of higher ovulation rates following the highest doses of gonadotrophin.

ACKNOWLEDGMENTS The authors wish to thank C. Muir and B. Thatcher for technical

assistance and C. Hawthorne and farm staff for attention to the animals.

Progesterone used was a gift of British Drug Houses Ltd. The project was aided in part by a University Grants Committee grant (64/215).

REFERENCES AVERILL, R. L. W. 1958: The production of living sheep eggs. 1. agric.

Sci. Camb. 50: 17-33. 1959: Ovulatory activity in mature Romney ewes in Otago.

N.Z. Jl agric. Res. 2: 575-83. ~----1965: Ovulatory activity in mature Romney ewes .in New

Zealand. N.Z. Jl agric. Res. 7: 514-24. AVERILL, R. L. W.; ROWSON, L. E. A. 1958: Ovum transfer in the

sheep. 1. Endocr. 16: 326-36. BERRY, R. 0.; SAVERY, H. P. 1958: A cytological study of the maturation

process of the ovum of the ewe during normal and induced ovulation. In "Reproduction and infertility 3rd Symp." pp. 75-82 Ed F. X. Gassner, London. New York, Paris, Los Angeles. Pergamon Press Ltd.

CUMMING, I. A.; McDoNAlD, M. F. 1967: A note on oestrous response to progesterone treatment in New Zealand Romney ewes. N.Z. Jl agric. Res. 10: 316-8.

DAVIES, H. L. 1960: Reduced fertility associated with the use of multiple injections of progesterone followed by pregnant mare serum. A list. vet. 1. 36: 20-3.

EDGAR, D. G. 1958: The induction of ovulation and oestrus in ewes during anoestrus. Proc. N.Z. Soc. Anim. Prod. 18: 97 (abstr.)

HANCOCK, J. L.; HOVELl., G. J. R. 1961: Transfer of sheep ova. 1. Reprod. Fert. 2: 295-306.

HUNTER, G. L.; ADAMS, C. E.; ROWSON, L. E. A. 1955: Inter-breed ovum transfer in sheep. 1. agric. Sci. Camb. 46: 143-9.

LAMBOURNE, L. J. 1955: Preliminary studies in the control of oestrus and ovulation in Romney ewes with progesterone and pregnant mare serum. N.Z. 1. Sci. Technol. 37A: 187-95.

LAMOND, D. R. 1964: Synchronization of ovarian cycles in sheep and cattle. Anim. Breed. Abstr. 32: 269-85.

McDoNALD, M. F. 1961: Studies of the response of the anoestrous ewe treated with progesterone and pregnant mare serum. 1. agric. Sci. Camb. 56: 397-406.

McDONALD, M. F.; CH'ANG, T. S. 1966: Variation in ovarian activity of Romney Marsh ewes. Proc. N.Z. Soc. Anim. Prod. 26: 98-106.

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RAESIDE, 1. I.; LAMOND, D. R. 1956: Effects of progesterone and pregnant mare serum (PMS) administration in the anoestrous ewe. I. Oestrus, ovarian changes, and gonadotrophic potency of the pituitary gland. Allst. I. agric. Res. 7: 591-600.

SHELmN, 1. N.; MOORE, N. W. 1966: Survival of fertilised eggs trans-ferred to ewes after progesterone treatment. I. Reprod. Ferr. 11: 149-51.

WALLACE, L. R. 1954: Studies in the augmentation of fertility of Romney ewes with pregnant mare serum. I. agric. Sci. Camb. 45: 60--79.

WALLACE, L. R.; LAM BOURNE, L. I.; SINCLAIR, D. P. 1954: Effect of pregnant mare serum on the reproductive performance of Romney ewes. N.Z. I. Sci. Technol. 35A: 421-36.

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