Ovine estrus synchronization and superovulation using norgestomet B and follicle stimulating hormone-pituitary

THERIOGENOLOGY

OVINE ESTRUS SYNCHRONIZATION AND SUPEROWLATION USING NORGESTOMET B AND FOLLICLE STIMULATING HORMONE-PITUITARY J. Ruttle, S. Lucero, S. Key, M. Daniels, F. Rodriguez and H. S. Yim Department of Animal and Range Sciences College of Agriculture and Home Economics New Mexico State University Las Cruces, NM 88003 Received

for publication: Accepted:

September 11, 1987 MaY 20, 1,988

ABSTRACT Finewooled Rambouillet range ewes were used in a study to determine the feasibility of using a progesterone ear implant to synchronize estrus. In addition, some of the ewes were further treated with injections of follicle stimulating hormone-pituitary (FSH-P) to induce superovulation. Five days following estrus detection and breeding, FSH-P-treated ewes were laparotomized and surgically flushed to recover embryos. The number of corpora lutea (CL), the total number of embryos and the number of transferable embryos recovered were recorded along with the number and size of follicles present on both ovaries. Ewes synchronized as recipients were laparotomized for surgical transfer of embryos 5 d following estrus. The number of CL and follicles were Response to superovulation by FSH-P did not differ (P>O.O5) recorded. between age groups of ewes when the number of CL present was counted. However, the total number of embryos flushed and good embryos was lower The number of follicles present (PC.O5) CL, than 3-, 4- or 7-yr-old ewes. Only slight variation was observed in the number of follicles in recipient ewes. Among donor ewes receiving FSH-P in addition to Synchro-Mate B, 71% were detected in estrus within 48 h of implant removal vs 55% of the recipients. Key words:

ovine,

synchronization,

superovulation,

Synchro-Mate

B

INTRODUCTION

the -

Estrus synchronization and embryo transfer n sheep has not received same attention as in cattle in the United States. While several

Acknowledgments Journal Article 1359, New Mexico Agricultural Experiment Station Rsssearch was supported by State Project 1-3-42177 and contributed to w-112. 4ppreciation is expressed to Diamond A Cattle Company, Roswell, NM; Bill Roach, Caprock, NM, who furnished sheep for the study; and to Dr. Ernie Henderson and Ceva Laboratories, Inc., Overland Park, KN, for furnishing the Synchro-Mate B implants.

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1988 VOL.

30 NO. 2

421

I’HERIOGENOLOGY

commerical

prostaglandin

F2CY products

are

available

for

bovine

estrus

synchronization, as well as a progesterone implant, none are commercially marketed for sheep in the United States. Recently, progesteroneimpregnated pessaries have been used to synchronize estrus in sheep (1, 2), following earlier efforts to orally administered progestins (3, 4). Further research using intravaginal pessaries along with gonadotropin injections (5, 6) indicated that ovine synchrony and fertility could be improved over using a progestin alone. The use of prostaglandin F2a has bee!: :.t.i” i,:.d in the ewe (71, and results have shown that, while synchrony was achieved, fertility of the resulting estrus was lower (P
objectives

of

this

study

were

to

1)

investigate

the

response

of

fine wool ewes to progestin implantations along with FSH-P injections to induce superovulation donor ewes in an embryo transfer program, 2) determine the suitability of ewes implanted with a progestin only (no gonadotropin) to serve as recipient ewes in an embryo transfer program. Parameters studied included ovulatory response, embryo yield in donors, follicular development and time of estrus following embryo quality, implant removal. MATERIALS

AND METHODS

Animals Finewool Rambouillet ewes from two New Mexico commercial sheep ranches were used in this study. Ewes ranged from 2 to 7 yr of age and had all lambed the previous year. No other reproductive history Ewes were selected as donors based on phenotypic was available. and an face factors, primarily wool quality, physical size open Forty-nine donor ewes were designated to receive both covering. synchronization and superovulation treatments ; 80 recipient ewes received only the synchronization treatment. Synchronization To

synchronize

and

Superovulation estrus,

both

donor

and

recipient

ewes

were

implanted

subcutaneously behind the ear (midwaya between base and tip) with an This size implant was obtained implant containing 3 mg of Norgestomet. by cutting a commercially sold 6-mg bovine implant, Synchro-Mate B, in Stage of the estrous cycle was not known at the time of two parts. which was done between October 15 and November 15, the implantat ion, normal breeding season for ewes in this area. Implants remained in time they were removed. Ewes designated as place for 12 d at which in a series of injections as follows : Day 10 donors received FSH-P following implantation, 4 mg FSH-P twice daily; Day 11, 3 mg FSH-P twice daily; and Day 12, 2 mg FSH-P twice daily (total = 18 mg). Recipients Ceva Laboratories, Inc., Burns-Biotec Laboratories,

422

Overland Inc.,

Park, Omaha,

KN. NE.

AUGUST

1988 VOL.

30 NO. 2

THERIOGENOLOGY

received

the 3 mg progestin implant only. Immediately following implant vasectomized rams equipped with marking harnesses were joined remova 1, with the recipient ewes to detect estrus. Records of ewes marked was obtained at 12-h intervals up to 60 h. Intact rams of known fertility and equipped with marking harnesses were joined with donor ewes, and the breeding activity was recorded up to 60 h post implant removal. Any not showing estrus within 60 h were removed from the group and ewes recorded as having no response. Five days following estrus, donor ewes were surgically flushed via midventral laparotomy, and embryos were transferred to recipients that were also 5 d post estrus. Flushing was accomplished by inserting sterile 3-mm plastic tubing midway between the bifurcation of the uterine horns and the utero-tubal junction of each horn. The plastic tubing was 5 cm in lenth and attached to a 50-ml syringe. Entry into the lumen was by puncture with a sterile, closed hemostat. The flushing medium was directed towards the oviduct, with the medium collected from the fimbria into a collection dish containing a 70-p filter,c Embryos were deposited into the recipient uterine horns approximately 4 cm frzm the utero-tubal junction by puncture with a sterile Pasteur pipet. Ovarian

and

Embryo

Measurements

During midventral laparotomy, the number of CL on each ovary of bot.h donor and recipient ewes was recorded. Follicles present on each ovary were measured and recorded as 2 mm, 3 to 4 mm and >4 mm. Donor ewes were flushed with Dulbecco’s phosphate buffered saline containing 2% heat-treated calf serum,e and examined immediately. The total number of embryos recovered was recorded, and the embryos were then examined for morphology. Embryos were classed as follows: 1) Excellent - perfectly symetrical even granulation, a well-defined distinct outline, no blastomere extrusion, zona pellucida intact; 2) Good - even granulation, distinct outline, some blastomere extrusion, zona well-defined a pe:.lucida intact, occasionally slightly asymetric in shape; 3) Fair integral, outline in extrusion hazy parts, some blas tomere and slightly asymrtric in shape, zona pellucida intact; 4) degeneration, Poor or Degenerate - uneven granulation, hazy out line, much blastomere extrusion and degeneration. Only embryos classed as Excellent or Good were considered suitable for transfer. Flushing at Day 5 post estrus resulted in the recovery of embryos that were from eight-cell blastomere to morula stages. No blas tocysts were recovered. Stat

is t ical Ovulatory

follicular va:riance Statistical response

Analyses response,

total

embryos,

transferable

embryos

and

measurements were analyzed by the least squares analysis of according to the General Linear Models procedures of the Analysis System (9). The effects of age of ewe on ovarian were analyzed within donor and recipient groups.

i

IMMUNO Systems Inc., Biddeford, ME. Fisher Scientific Company, Dallas, TX. e .DIFCO Laboratories, Grand Island, NY.

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1988 VOL.

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423

11

8

7

4

5

6

5.1 -+ 1.4Cd 1.7 -+ 0.9d

14.2 -+ 0.9'

7.4 -+ 1.6'

12.8 -+ 2.3'

to

treatment

1.7 -+ 0.9d

5.1 2 1.4Cd

5.9 -+ 1.8Cd

7.4 -+ 2.2c

Transferable Embryos

8.0 -+ 1.5'

7.5 -+ 2.3'

1488 -+ 1.9'

Total Embryos

ees a,%

B

and

1.7 + o.7c

2.5 2 0.8'

3.8 + 1.4'

10

0.4 -+ o.2c

1.8 -+ 0.4d

Days

1.0 -+ 0.4'

1.5 -+ o.5c

5.9 -+ 1.3c 3.7 -+ l.lCd

1.0 _+ 0.3'

>4 mm

follicle

3.8 + 0.5Cd

Follicles measured 3-4 mm

Norgestomet

1.7 + 0.6'

2 mm

with

Treatment was 3.0-mg Norgestomet implant for 12 d plus 18 mg FSH-P given between and 12. b c d Values are mean + SEM. ’ Within columns, means with no superscript letter in common differ (~<0.05).

a

7

3

Corpora Lutea

Ovarian response of donor stimulating hormone-pituitary

age (years) number

Ewe

Table 1.

THERIOGENOLOGY

RESULTS Done r Response Ovulatory response of 33 donor ewes was determined by the number of CL observed on the ovaries at laparotomy, which ranged from a mea” of 8.0 for 5-yr-old ewes to a mean of 14.2 for ‘I-yr-old ewes (Table 1). There was no difference between age groups (P>O.O5) in ovulatory response. However, total embryos recovered and transferable embryos Ewes in the 3-, 4- and 5-yr-old dif iiered (PO.OS) in the number of 2-mm fol Licles and >4-mm follicles. The 6-yr-old ewes produced fewer (P
Recipient

Response

Recipient ewes received only Ovulation response (CL) and follicles Table

2.

Ovar ia” response Synchro-Mate Ba$b

of

a

3-mg progestin implant for measured are shown in Table

recipient

ewes

to

12 2.

treatment

d.

with

Ewe age (years)

2 3 4 5 6 7

Number

57 10 12 2 6

Follicles

Corpora lutes

1.6 1.5 1.4 1.6 2.5 1.1

5+ T -c + s

2 mm

0.2cd 0.2c 0.1c O.lcd 0.5d O.lC

1.2 1.4 5+ 0.9cd 0.8c 2.0 5 0.6cd 4.0 T 0.8d 0.5 T 0.5cd 4.3 5 l.ld

Treatment was 3.0-mg Norgestomet c ti Values are mea” + SEM. ’ Within columns, -means with no (P
it

implant superscript

2.8 1.2 1.2 3.5 3.0 3.0 for

measured 3 4 mm

++ T; 5 ,. i

0.8cd l.lcd 0.3c 0.8d 1.ocd 1.1cd

>4 nun

o-0 0.7 0.9 0.9 1.0 1.5

+ -c -c + _:

o.icd 0.3cd 0.2cd o.ocd 0.4d

12 d. letter

in

common differ

Only small differences were observed in ovulatory response among 42 recipient ewes that were laparotomized. Mea” numbers of CL ranged from with least number of CL observed among 7-yr-old 1.1 to 2.5, ewes. Follicles 2 mm in size were more numerous (P0.05). 4-yr-old ewes. Few recipient ewes had follicles measuring >4 mm at time of laparotomy, and no important differences were observed.

AUGUST 1988 VOL. 30 NO. 2

425

THERIOGENOLOGY

Occurance A

of

Estrus

critical

part

synchronization recipient ewes Table

3.

of are

Estrus

of embryo transfer any Times of detected estrus estrus. shown below (Table 3).

occurence

in

Syncro-Mate Hours

Donors, No. Recipients,

(X) No.

(X)

12

24

3(6) 0

3(6) l(1)

to

B treated

estrus

after

36 0

t 3-mg Norgestomet implant for 12 d. c Donors received 18 mg FSH-P between Days Recipients received 3-mg implant only.

both

implant 60

35(71)

0

44(55)

19(24)

10

and

is

the

donor

and

ewesa’bac

48

ll(14)

program in

removal No response 8( 16) 5(6)

12.

that received gonadotropin injections during three ewes (6%) were in estrus 12 h following implant removal, and an additional three were detected in estrus at 24 No additional ewes came into estrus at 36 h. However, h. considerable estrual activity occurred at 48 h following implant removal, when 35 (71%) ewes were detected in estrus. No additional ewes came into estrus and eight show estrus. at 60 h post removal, ewes (16%) failed to Recipients were not stimulated by exogenous gonadotropins, and only one Eleven ewe among 80 implanted with progestin showed estrus at 24 h. ewes (14%) were in estrus at 36 h post removal and, as in the donors, the greatest activity was at 48 h, when 44 (55%) of the recipient ewes were in estrus. Estrual activity among recipients was still occurring as late as 60 h post implant removal, with 19 ewes (24%) observed in estrus. only five (6%) failed to respond to Among all recipients, progestin treatment. the

.Among 49 donor ewes implantat ion period,

DISCUSSION

Our study shows that a 3-mg progestin implant induces estrus and offers a reliable synchronization method. Ovarian response in ewes has indicated that using a 3-mg progestin implant and 18-mg FSH-P injection superovulation can be achieved in ewes ranging in age from 3 to 6 yr of the total number of embryos However, recovered may be lower in age. older ewes. Follicular development observed 5 d post treatment appeared to be essentially the same for all ages, consisting mostly of follicles <4 mm in diameter. Response of recipient ewes to treatment with only 3 mg of progestin Ovulatory rates all exceeded 1.0, suggesting that synwas excellent. chronization treatment did not interfere with normal ovarian function, and that this treatment would be useful for natural service breeding programs as well as for preparing recipients for embryo transfer. The uterine environment in the synchronized recipient was evidently normal f because more than 60% of these recipient ewes subsequently lambed to f

Unpublished Cruces, NM.

data,

J.

Ruttle,

NM Agricultural

AUGUST

Experiment

1988 VOL.

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30 NO. 2

THERIOGENOLOGY

embryo transfer,f Synchronization of estrus was satisfactory with the majority of donor (71%) and recipient (55%) ewes in estrus at 48 h, which closely follows patterns observed in the bovine (10). This response resulted in only a 12-h asynchrony between donor and recipient at the time of transfer, which is well within the maximum 24-h range According to Moore, recipients suggested by Moore (11) for the ewe. but when donors and recipiwill support embryos 2 d older or younger, ents are 3 or more days out of synchrony, very few embryos survive transfer. Results indicate the protocol reliable means of ovine synchronization

described in this and superovulation,

study

offers

a

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Waite, A. B. and fertility

Some effects of in the ewe.

progesterone J. Anim.

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2.

Curl, W. E., Bogard, G., Cockrell, T. and Synchronization of estrus through the use of progestin. Livestock and Feeders Day Proceedings, (1966). College. pp. 117-122

3.

Hogue, D., Hansel, W. and Bratton, R. W. Fertility of ewes naturally and artificially after estrous cycle synchronization an oral progestational agent. J. Anim, Sci. -21:625-627 (1962).

4.

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5.

Reproductive performance of G. M. Ruttle, J. and Southward, treated with flurogestone acetate and gonadotropins. N.M. Exp. Sta. Res. Report m:l-4 (1979).

Hudson, F. intravaginal Texas Tech

bred with

ewes Agr .

6.

Lamb production from Ruttle, J., Kott, R. and Southward, G. M. finewool range ewes synchronized with progestin pessaries and FSH-LH injections. N.M. Ag. Exp. Sta. Res. Report -431:1-3 (1981).

7.

Ruttle,

J.

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and

Bredee,

F2a.

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N.M. Ag.

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Sta.

Bul.

in

-719:1-18

ewes

with

(1986).

8.

Lucero, S., Ruttle, J., Key, S., Rodriguez, F. and Yim, H. S. Ovulatory response by finewool ewes to PMSG or FSH-P. Proceedings, West. Sec. Amer. Sot. Anim. Sci. (1986). -37:23-25

9.

SAS. Inc.

SAS

Cary,

User’s

Guide.

Statistical

10.

Ruttle, J., Ramirez, A., Archunde, synchronization in beef heifers with Sta. Res . Rpt . -381:1-4 (1978).

11.

Adams, Florida,

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C.

E. 1982,

Analysis

System

Institute,

NC, 1979.

Mammalian Egg Transfer. pp. 119-133.

1988 VOL.

30 NO. 2

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CRC Press

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Inc .,

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427