Field evaluation of a technique for transcervical intrauterine insemination of ewes

THERIOGENOLOGY FIELD EVALUATION OF A TECHNIQUE FOR TRANSCERVICAL INTRAUTERINE INSEMINATION OF EWES G.W. Halbert, H. Dobson,' J.S. Walton,3 P. Sharpe4 and B.C. Buckrelll IDepartment of Population Medicine 2Department of Clinical Studies Ontario Veterinary College 3Department of Animal and Poultry Science University of Guelph Canada NlG 2Wl Guelph, Ontario 4 New Liskeard College of Agricultural Technology New Liskeard, Ontario Canada Received

for publication: August 18, 1989 Accepted: March 13, 1990

ABSTRACT In this study, three groups of ewes were inseminated with a transcervical intrauterine technique in order to evaluate the for a commercial suitability of the artificial technique insemination (AI) program using frozen semen. The ewes were _ synchronized into estrus with vaginal pessaries and pregnant mare serum gonadotrophin (PMSG) and were inseminated between 48 and 55 h following pessary removal. In Flock I, maiden and multiparous ewes (n = 12) received fresh semen containing 400 X 40 106 spermatozoa. Flock divided accordingly: II was multiparous ewes were inseminated with frozen semen using the transcervical ewes were technique and 40 inseminated laparoscopically with frozen semen. In Flock III, 38 multiparous ewes were inseminated transcervically with semen frozen Pregnancy diagnosis was based containing 150 X lo6 spermatozoa. on blood progesterone analysis 18 d following breeding, and real time ultrasound examination at 40 d. Pregnancy rates were 50, 50 respectively, the three groups inseminated and 68%, for transcervically the ewes inseminated and 70% for laparoscopically. The lambing rates were 50, 55 and 40%, for the ewes inseminated transcervically and 65% respectively, for the ewes inseminated laparoscopically. Lambing results were confirmed by matching the breeding dates to the lambing dates. that the described These results demonstrate technique is suitable for further evaluation using frozen semen in a commercial sheep AI program. Key words: transcervical,

intrauterine

insemination,

trial ewes

Acknowledgment The authors wish to thank the Ontario Ministry Agriculture and Food for the financial support of this study.

JUNE 1990 VOL. 33 NO. 6

of

1231

THERIOGENOLOGY INTRODUCTION The success of artificial insemination [AI) programs is based on factors which are specific to the ewe, the ram and the insemination technique. Pregnancy rates and lambing results are used to evaluate AI programs. Pregnancy rates from insemination with fresh semen can be similar to those obtained with natural breeding when semen is deposited in the cervix or within the uterus. However, frozen semen must be deposited within the uterus to obtain acceptable pregnancy rates (1). Fresh chilled semen can only be stored for 24 h following collection before fertilization rates are reduced (2), whereas frozen semen can be stored indefinitely, increasing the potential benefits of AI. There has been limited understanding of the efficacy of freezing and thawing ram semen, but a growing interest in the technology is evident. Recent studies report the potential for excellent post-thaw fertility (3,4). Due to surgical requirements the laparoscopic technique of intrauterine insemination is not suitable for routine use in many commercial programs. Eccentrically located rings within the cervical canal and ring small openings prevent consistent intrauterine insemination, as is routinely performed in other domestic ruminants (6,7). A technique for transcervical passage has been reported (8) and modified by others (9) but has not been adapted to a commercial program. Preliminary pregnancy rate results with these techniques on ewes in natural estrus and using frozen semen ranged from 69 to 83%. This study was designed to evaluate the success of a technique for transcervical intrauterine insemination and to determine if it is suitable for a commercial breeding program. MATERIALS

AND METHODS

General Three groups of ewes from different flocks were inseminated during the fall of 1988. The ewes in each group were chosen based on availability, with no selection for age, parity or Each ewe received a vaginal pessary containing 40 mg breed. fluorogestone acetate (Chronogest),a which was removed 14 d later when 500 IU of pregnant mare serum gonadotrophin (PMSG; Equinex)b was administered intramuscularly.

aIntervet West Hill, Canada. bAyerst Laboratories, Montreal,

1232

Quebec, Canada.

JUNE 1990 VOL. 33 NO. 6

THERIOGENOLOGY

Group I Twelve Suffolk ewes, of which seven were multiparous and five were maidens, from the University of Guelph flock, were Inseminations began synchronized into estrus as described above. 55 h following pessary removal. Each ewe was led into a Commodore trimming cradleC and the arms of the cradle were allow rotation tightened around ewe to into dorsal the recumbency. The hindquarters of the ewe were elevated 30 to 40° by lifting the end of the cradle onto a chair, and a modified Grave's duskbill speculumd was introduced into the vagina. The external cervical OS was located within the vagina and Bozman 1O.7511 forcepse were fastened to the vaginal tissue surrounding it. The forceps were pulled back to retract the cervix into the vaginal canal within the speculum. An insemination instrument (10) was introduced into the external OS through the opening of the speculum, and was manipulated through the canal into the deposited. If the instrument was uterus where semen was obstructed and could not be advanced within the canal, the semen Resistance to the instrument was was deposited at thatlocation. reduced as it passed through each ring, and, when it reached the The relative uterus, the resistance was almost eliminated. location of the instrument within the canal was determined and Locations of recorded by counting rings through which it passed. insemination were recorded as uterine, deep cervical and mid Problems associated cervical, which includes the cervical OS. with the inseminations were recorded. The semen had been collected from a Suffolk ram at United Breeders Inc. on the morning of the insemination day and was It was stored at 15OC for extended with a skim milk extenderf. approximately 3 h when 400 x IO6 spermatozoa were inseminated into each ewe by injecting the semen through the insemination instrument. All ewes were confined for 18 h after breeding before being Eighteen days following returned to their normal housing. insemination, a blood sample was collected from the jugular vein and the plasma was analyzed for progesterone by radioimmunoassay Ewes with progesterone levels of 1 ng/ml or higher were (11). Sixty days post breeding the ewes recorded as being pregnant. real-time were examined array for pregnancy with a linear transducer (SSD-210DX)g. ultrasound machine with a 5 mHz Pregnancy and lambing results were analyzed and compared.

CPolvendale Livestock Feeding and Handling Equipment, England. dWelch Allyn, Mississauga, Ontario, Canada. eWitte GMBE, D5650 Solingen II, West Germany. fUnited Breeders Inc., Guelph, Ontario, Canada. gAloka, Tokyo, Japan.

JUNE 1990 VOL. 33 NO. 6

London,

1233

THERIOGENOLOGY Group II Eighty multiparous Suffolk ewes at the New Liskeard College of Agricultural Technology flock were synchronized into estrus in two groups, with one week between the insemination of each group. Forty ewes were inseminated each day 48 to 54h after pessary removal. The ewes were assigned in a systematic manner so that 20 were inseminated using a laparoscopic technique (12) and 20 were inseminated using the transcervical technique as described In each insemination technique, each ewe for Group I ewes (10). received 10 mg xylazine (Rompun)h intramuscularly approximately 5 The ewes that were inseminated min prior to insemination. of the udder and laparoscopically were clipped in front surgically prepared. They were inseminated with 0.25 ml of semen into each uterine horn, whereas the ewes bred transcervically received two 0.25-ml straws at the location in the reproductive tract that the insemination instrument reached. The straws were attached to the insemination instrument and the semen was forced through the instrument by air pressure. The relative location of insemination was recorded, as were any problems or abnormalities. The semen was collected from a Dorset ram by United Breeders Inc., with each 0.25-ml straw containing 75 x lo6 spermatozoa. The extender used .was a commercially available bovine semen Triladyl)i. extender The extended semen was then chilled from 35OC to 4 6 C in one step over a 2-h and then frozen in an automatic freezer (Digit Cool 5300). !ieriod, When the semen was needed, it was thawed in a water bath at 35OC for 20 sec. After insemination, the ewes were confined for 12 h before being returned to normal activity. Twelve days later, four Suffolk rams were introduced to the group of ewes to breed those which had returned to estrus. Eighteen days after insemination, blood was collected from the jugular vein of the ewes for determination of plasma progesterone by radioimmunoassay. Ewes with 1 ng/ml or higher of plasma progesteron? were recorded as being pregnant. A linear array ultrasound machine with a 5 mHz transducer was used 60 d following insemination to examine the ewes for pregnancy. The examiners did not differentiate between pregnancies resulting from the artificial insemination or subsequent natural breeding. All ewes were housed and managed in the same manner until lambing. The ewes that lambed and the number of lambs born per ewe were recorded, analyzed and compared. The lambs born from the artificial breeding were differentiated from the natural breeding by the lambing date and color of the offspring.

hHaver Bayvet, Etobicoke, Ontario, Canada. laini Tube of America Inc., Cambridge, Iowa, USA. jIMv International Corp., L'Aigle, France.

1234

JUNE 1990 VOL. 33 NO. 6

THERIOGENOLOGY

Group III Thirty-eight crossbred white-faced ewes, with an average condition score of three on a scale of one to five, were selected from a commercial flock for breeding. Inseminations began at 53 h and were completed by 60 h following pessary removal. The ewes were inseminated in dorsal recumbency by the technique described for Group I (10); each ewe received two 0.25-ml straws of Dorset semen which had been collected! processed and inseminated as described for Group II. The relative location of insemination was recorded as well as any problems or abnormalities encountered. The ewes were confined for 12 h following breeding and then mixed with a larger group of ewes that were on a rising plane of nutrition. Beginning 12 d after AI, rams were introduced into the group to breed ewes which came into estrus. Forty days following AI, the ewes were examined for pregnancy with a linear array realtime ultrasound machine with a 5 mHz transducer. Determination of the duration of pregnancy from the ultrasonic image was not made. The lambs born from AI could be differentiated from the natural breeding lambs based on lambing date. The ewes lambing and the number of lambs born were recorded, analyzed and compared. Data Analysis For all three groups, the pregnancy and lambing rates, mean number of lambs born per ewe bred and per ewe lambing, and standard err rs were calculated by the Statistical Analysis R A Chi-square analysis was used to analyze the System (SAS). difference at P < 0.05 in pregnancy rates on Day 18 between the ewes bred laparoscopically and those bred transcervically in Group II. RESULTS Group I All of the ewes were difficult to restrain in the trimming cradle because their hind legs were not contained by the arms of the cradle. The vaginal wall of six of the ewes folded inbetween the upper and lower bills of the speculum, which limited the field of vision within the speculum. In the ewe lambs, the speculum could not be opened as wide as in the mature ewes, which made finding the cervix more difficult and provided less working space through which the grasping forceps and inseminating instrument could be manipulated. from Day-18 plasma The rates determined pregnancy to the ones progesterone concentrations were equivalent kSAS Institute Inc., Cary, NC, USA.

JUNE 1990 VOL. 33 NO. 6

1236

THERIOGENOLOGY

determined by the ultrasound examination (Table 1). Two ewes diagnosed pregnant on Day-18 were not considered pregnant on Day-60 and did not lamb (Tables 1 and 2). Two ewes diagnosed not pregnant on Day 18 were diagnosed pregnant on Day 60 and did lamb (Tables 1 and 2). Pregnancy rates were higher when the semen was recorded as being deposited within the uterus, rather than within the cervix (Table 1). From the deep cervical to the mid cervical location, The mean number of the pregnancy rates decreased (Table 1). lambs born per ewe shows the same trend of decreasing from the uterine to mid cervical locations (Table 2). Group II The vaginal wall of 10 ewes folded between the upper and lower arms of the speculum and partially obscured the opening This made locating the cervix and within the speculum. manipulating the instruments within the vagina difficult. The pregnancy rate from the progesterone test was not significantly different (P -C 0.05) between the ewes bred and those bred transcervically (Table 3; laparoscopically x2 = 2.55). However, the ultrasound results for the ewes bred transcervically were higher than for ewes bred laparoscopically (Table 3). The lambing rates were marginally higher for the ewes bred laparoscopically (Table 3). There was one ewe that was bred laparoscopically and which was diagnosed pregnant based on the progesterone test which did not lamb. Two ewes bred transcervically and diagnosed not pregnant by the progesterone test did lamb. The mean number of lambs born per ewe was lower for those inseminated with the transcervical method (Table 3). For the ewes bred by the transcervical technique, the Day-18 pregnancy rate (Table 1) and lambing rate (Table 2) were higher when insemination was in the uterus compared with the cervix, where rates were higher for the deep cervical than mid cervical location (Tables 1 and 2). By the Day-60 ultrasound examination, all the ewes were pregnant (Table l), and the lambing rate was 5% higher than the pregnancy rate on Day 18 (Tables 1 and 2). For the ewes bred laparoscopically, not all were diagnosed pregnant from the ultrasound examination, and the lambing rate was 5% lower than the pregnancy rate on Day 18 (Table 3). Group III Ewes were fully restrained in the trimming cradle, and there was no problem with folds of the vaginal wall obscuring the opening within the speculum. Locating the external cervical OS and manipulating the inseminating instrument within the cervical canal in general was easier than with the other two groups.

1236

JUNE 1990 VOL. 33 NO. 6

t

$ &I

E

m

4

6

Deep cervical

Hid cervical

a400 x 10b spermatozoa examination - Day 60. b150 x lo6 spermatozoa examination, Day 60. c15O x lo6 spermatozoa

12

2

Total

rates

Ewes inseminated (n)

1. Pregnancy

Uterine

Location

Table

Ia

location

Suffolk crossbred

- frozen;

Suffolk

50

17

75

100

Day 60 %

of

- frozen;

- fresh;

50

17

75

100

Day 18 %

Ewes pregnant

Group

for

ewes;

radioimmunoassay

radioimmunoassay

for

for

progesterone, examination,

plasma

progesterone

38

100

plasma

6

3

29

(n)

Ewes inseminated

Day 18;

- Day 18;

ultrasound

ultrasound

68

33

33

79

%

Ewes pregnant Day 40

IIIc

technique

Day 40.

Group

intrauterine

100

100

100

Day 60 90

ultrasound

50

40

31

83

11

white-faced

ewes;

ewes;

transcervical

Ewes pregnant

IIb

the

Day 18 %

Group

using

9

13

18

Ewes inseminated (n)

insemination

m

K

2 B

g

4

6

12

Deep cervical

Mid cervical

Total

a400 x lob spermatozoa examination - Day 60. b150 x lo6 spermatozoa examination, Day 60. c15O x IO6 spermatozoa

2

Uterine

Location

Ewes inseminated (n)

Suffolk crossbred

- frozen;

ewes;

radioimmunoassay

white-faced

ewes;

55

22

39

83

radioimmunoassay

40

9

13

18

Ewes lambing %

error

ultrasound

examination,

for plasma progesterone,

progesterone

38

6

3

29

of

insemination

?O

0.8 !. 0.2

0

0

1.1 f 0.2

Lambs born /ewe inseminated

Day 40.

Day 18; ultrasound

- Day 18; ultrasound

45

0

0

59

Ewes lambing %

Group IIIc

location

Ewes inseminated (n)

(SEH) for

for plasma

1.2 t 0.2

0.4 f 0.3

0.8 f 0.3

1.8 f 0.2

Lambs born /ewe inseminated

Group IIb

per ewe f standard technique

Ewes inseminated (n)

ewes;

- frozen;

Suffolk

1.2 !: 0.4

0.3 + 0.3

1.8 + 0.6

2.5 ? 0.5

Lambs born /ewe inseminated

- fresh;

50

17

75

100

Ewes lambing %

Group Ia

Table 2. Lambing rates and mean lambs born using the transcervical intrauterine

THERIOGENOLOGY

JUNE 1990 VOL. 33 NO. 6

1239

THERIOGENOLOGY The pregnancy rate when the semen was deposited in the in the cervix uterus was higher than when it was deposited There was no difference between pregnancy rates (Table 1). resulting from semen deposited into the deep cervical and mid cervical region (Table 1). The ewes which lambed had all been There were nine ewes inseminated within the uterus (Table 2). diagnosed pregnant from the ultrasound examination that did not lamb that had been artificially inseminated and there were two that were artificially inseminated ewes which lambed not diagnosed pregnant. DISCUSSION To be attractive to producers, an AI program using frozen semen should result in pregnancy rates that are similar to those obtained in other domestic species and should provide opportunity to use rams that the producers do not normally have access to in a natural breeding program. Among the problems encountered in our study were that the large Suffolk ewes were harder to restrain than the crossbred white-faced ewes, and there was difficulty with the vaginal walls folding around the speculum. The larger ewes have larger vaginas and the speculum did not open adequately to stretch the vaginal walls. This was a significant problem for locating the external the cervix instruments within OS of and manipulating the speculum. Modifications to the speculum to allow it to open further and widening the top and bottom extensions to open the vaginal cavity more freely are needed to correct this problem. In Group I the problems encountered with restraint and manipulation of the insemination instrument through the cervical canal may have limited the success rate with fresh semen to result in a pregnancy rate of 50%. Also, 42% of the group were maiden ewes, had smaller vaginas that resulted in more difficulty locating the external cervical OS. The laparoscopic technique was used in the second group to act as a standard for intrauterine insemination on which to compare the results of the transcervical technique, since results using laparoscopic intrauterine insemination are well documented. The management, housing, nutrition and number of spermatozoa used were the same for all ewes bred, whether transcervically or laparoscopically. Based on the Day-la pregnancy rates there was no significant difference between from the results either insemination technique used (P < 0.05). In the ewes bred laparoscopically, the difference in the Day-la pregnancy rate and the lambing rate could be due to either early embryonic death occurring after the 18th d of pregnancy, or a false positive interpretation of the progesterone level, which occurs when progesterone is elevated but the animal is not pregnant. This occurs with early embryonic death between Days 14

1240

JUNE 1990 VOL. 33 NO. 6

THERIOGENOLOGY and 18 of the cycle, insemination occurs.

or

if

the

ewe

is

not

in

estrus

when

There was no instance of early embryonic death in ewes inseminated transcervically beyond Day 18. In fact, there were two false negative interpretations of the progesterone test which resulted in the lambing rate being higher than the pregnancy rate. False negative results are likely due to laboratory error or to the cut point of 1 ng/ml of plasma progesterone being too high. TWO ewes had plasma progesterone concentration below 1 ng/ml but were pregnant. The pregnancy rate in the third group was higher than observed in Groups I and II, but there were many false positives as a result of failure to differentiate pregnancies resulting from breeding subsequent to AI. Early embryonic death occurring just prior to ultrasound examination may account for some of these results as well. The false negatives resulted from the inability of the examiners to diagnose pregnancy from the ultrasonic image. The lambing rate in the third group was lower than in the other groups (Table 2) and not as good as expected based on the increased success of intrauterine deposition of the spermatozoa. Each group was inseminated with semen from a different ram, which could result in the pregnancy and lambing rates being different between the groups. One major management factor that was different with Group III was that lambs had been weaned from the ewes inseminated only three weeks previously. This may have negatively affected the ovulation rate of the ewes and conception within Group III. In all ewes bred transcervically, when the semen was deposited within the uterus, the pregnancy rates were higher than when it was deposited in the cervix (Table 1). It also appears that pregnancy rates decrease the more caudal in the cervix that with findings the semen is deposited. This is consistent It cannot be concluded that reported by Andersen et al. (a). this trend is due to the actual location of insemination because it may be related to the reasons which prevent insemination beyond a specific location. A study in which semen would be deposited at specific locations along the cervix and in the uterus in an equal number of ewes is needed to establish this association. can benefit from AI The North American sheep industry with the current level of producer programs using frozen semen. interest, a well-designed commercial program could be successful. needs to be labour efficient and Animal restraint equipment The design of the adaptable to various management conditions. Commodore trimming cradle works well for getting ewes into dorsal elevating the recumbency, for mechanically but a method Modifications to the hindquarters of the ewe is required. speculum, making it adaptable to various size vaginas, would

JUNE 1990 VOL. 33 NO. 6

1241

THERIOGENOLOGY reduce the time required to locate the cervix. With the current limiting passage through the understanding of the reasons cervical canal (6,7) it seems that a different design for an insemination instrument would not improve the rate of cervical It must be realized by sheep industry producers and passage. intrauterine insemination is inseminators that although desirable, it is not likely to be possible with all ewes. A time limit, of perhaps 5 min. should be established for the manipulation to maintain efficient use of time as well as to prevent unnecessary trauma to the cervical canal. More studies of the affecting conception factors are needed to improve overall results of AI programs. For example, the optimal time for inseminating synchronized ewes as well as, the best extender for ram semen and the optimal number of spermatozoa per insemination are not known. Educating producers to maximize conception with proper nutrition and flock health management is likewise important. The transcervical intrauterine technique for insemination with frozen semen used in this study is suitable for a commercial program for sheep. The results of this study indicate that pregnancy and lambing results can be achieved which are consistent with those of frozen semen programs in other industries and with other techniques such as laparoscopic insemination of sheep. REFERENCES 1.

Evans, G. and Maxwell, insemination of sheep and 1987c, pp. 162-163.

2.

Langford, G.A., Marcus, G.J., Hackett, A.J.., Ainsworth, L., Wolynetz, M.S. and Peters, H.F. A comparison of fresh and frozen semen in the insemination of confined sheep. Can. J. Anim. Sci. =:685-691 (1979).

3.

Fiser, P.S., Ainsworth, L. and Fairfull, R.W. Cryosurvival of ram spermatozoa in hypertonic and isotonic diluents. Can. J. Anim. Sci. &Z:425-428 (1982).

4.

Ali, S.B.A. and Tischner, M. Freezing ram semen in aluminum packets and deep cervical insemination of ewes with a modified pipette. 11th Inter. Congress on Anim. Reprod. and AI. Vol. II, No. 219 (1988).

5.

Gustafsson, ram semen.

6.

More, J. Anatomy and histology of the cervix uteri ewe: new insights. Acta Anat. m:156-159 (1984).

1242

W.M.C. goats.

Salamon's artificial Hutterworths, Sydney,

B.K. Aspects of fertility with Cryobiology =:358-361 (1978).

frozen-thawed of the

JUNE 1990 VOL. 33 NO. 6

THERIOGENOLOGY

7.

Halbert, G.W., Walton, J.S., Dobson, H. and Buckrell, The structure of cervical canal of the the Theriogenology. Accepted for publication.

8.

Intrauterine Andersen, V.K., Aamdal, J. and Fougner, J.A. and deep cervical insemination with frozen semen in sheep. Zuchthygiene 8:113-118 (1973).

9.

Fukui, Y. and Roberts, E.M. Further studies on non-surgical intrauterine technique for artificial insemination in the ewe. Theriogenology m:381-393 (1978).

10.

Halbert, G.W., Walton, J.S., Dobson, H. and Buckrell, B.C. A technique for transcervical intrauterine insemination of ewes. Theriogenology. Accepted for publication.

11.

de Boer, G., Etches, R.J. and Walton, J.S. a solid-phase Can. J. radioimmunoassay for progesterone in bovine plasma. Anim. Sci. =:783-786 (1980).

12.

Killen, I.D. and Caffery, G.J. with the aid of a laparoscope.

13.

Stout, J-D. The role programs. Bovine Pratt.

14.

results of non-surgical Lawrenz, R. Preliminary intrauterine insemination of sheep with frozen-thawed semen. x:61-63 (1985). J. S. African Vet. Assoc.

15.

Sperm death and Hawk, H.W., Cooper, B.S. and Pursel, V.G. inhibition of sperm transport in the cervix of ewes in estrus after removal of the corpus luteum-bearing ovary. J. Anim. Sci. x:611-617 (1981).

16.

Hunter, R.H.F. and Nichol, R. Sperm transport, activation in the female reproductive tract. Sheep Vet. Sot. 9:64-68 (1985).

JUNE 1990 VOL. 33 NO. 6

Uterine insemination Aust. Vet. J. 2:95

of DHI u:31-38

records (1978).

in

herd

B.C. ewe.

of ewes (1982). health

storage and Ann. Proc.

1243