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Induction of ovulation and fertilization in the 90-day-old ewe lamb
THERIOGENOLOGY
INDUCTION OF OVULATION AND FERTILIZATION IN THE 90-DAY-OLD EWE LAMBta) T. P. Goerkefb) and R. H. Dutt Department of Animal Sciences, University of Kentucky Lexington, Kentucky 40506 (Received for publication:
October 20, 1975)
ABSTRACT The induction of ovulation and fertilization was studied in 50 sexually immature crossbred ewe lambs which received 500 or 1000 i.u. PMSG with or without pretreatment with progesterone. Pretreatment with progesterone did not significantly affect ovulation, fertilization or ova recovery rates. Also, progesterone pretreatment did not significantly affect weight of the ovaries, corpora lutea, follicular fluid or the reproductive tract. Lambs receiving 1000 i.u. PMSG had a significantly (PC.05) higher ovulation rate (13.2) than lambs receiving 500 i.u. PMSG (3.4). Weights of the ovaries, corpora lutea, follicular fluid and the reproductive tract were significantly (P1.05) higher in ewes receiving 1000 i.u. PMSG. None of the lambs exhibited estrus when checked daily following HCG injection. The low ova recovery rate (16 to 38 percent) was postulated to be due to failure of the fimbria of the infundibulum to surround the enlarged stimulated ovary and pick up released ova. INTRODUCTION Induction of ovulation, fertilization and successful ova recovery in the sexually immature female , if followed by ova transfer techniques, could result in shortening the generation interval and thereby enhance genetic improvement of livestock. Ovulation has been induced in the calf (1,2,3) and in the sexually immature ewe lamb (4,5). However, information is scanty on fertility and on the recovery rates of ova in the immature sheep. The present study was initiated to determine ovulation, fertilization and ova recovery rates in sexually immature ewe lambs following hormonal induction of ovulation.
(a) Published with approval of the Director of the University of Kentucky Agricultural Experiment Station as Journal Article 74-s-139. (b)Present address: Department of Animal and Food Sciences, University of Wisconsin, River Falls, Wisconsin 54022.
DECEMBER 1975
VOL. 4 NO. 6
195
THERIOGENOLOGY MATERIALS AND METHODS -Fifty sexually immature crossbred (Southdown x Western) ewe lambs, averaging 90 days of age (range 83 to 103 days) and 24 kg in weight, were randomly allotted on July 1 to 5 treatment croups of 10 lambs each. Prior to treatment lambs were maintained on a diet of high quality alfalfa hay and shelled corn. Ovulation was induced by subcutaneous injection (SC11 of Pregnant Mare Serum Gonadotropin (PMSG) at two levels (500 or 1000 i.u.) followed 2 days later with an intramuscular injection of 500 i.u. of Human Chorionic Gonadotropin (HCG). The effect of pretreatment with 25 mp of progesterone (four SC1 at d-day intervals) was also studied. Ten ewe lambs were slaughtered for control data and, in a factorial design, the remaininp 40 lambs were used to determine the effects of progesterone pretreatment at two levels of PMSG. All lambs were artificially inseminated on the day of, and one day following HCG injection, with 1 ml of freshly collected ram semen diluted 1 to 10 with previously heated whole milk. Lambs were checked for estrus daily with aproned teaser rams following HCG injection. The lambs were slaughtered 5 days after HCG injection. Immediately following slaughter the reproductive tracts were removed from the carcass and the number of corpora lutea counted to estimate ovulation rate. Ova were recovered from the oviduct and uterus by flushing the contents into a watch glass with 0.85% saline. Following recovery, ova were stained and examined at 430X for evidence of cleavage and the presence of spermatozoa in the zona pellucida. After ovaries were dissected free and weighed, the corpora lutea were dissected from the ovaries and weighed. The remaining portion of the ovaries were cut into l-mm slices which were blotted free of fluid with a paper towel. The slices were then weighed and the follicular fluid weipht determined by difference. RESULTS AND DISCUSSION Ovaries from control lambs were immature, with an average weight varying from 7 to 26% of that of ovaries from treated lambs (Table I). None of the lambs in the control group ovulated whereas all treated lambs did. The mean ovulation rate (9.1) in progesterone-pretreated lambs was higher but not sipnificantly different from the ovulation rate (7.5) in lambs not pretreated with progesterone. Percent ova recovered and percent ova fertilized were 37.7 and 4.6, respectively, for the progesterone-pretreated lambs and 20.9 and 4.0, respectively, for the non-pretreated lambs. Although both ova recovery and percent ova fertilized were slightly higher in the progesterone-pretreated lambs, differences due to progesterone treatment were not statistically significant. At the onset of the study it was postulated that progesterone might improve the uterine environment and result in higher fertility. However, this was not the case. These data are not in agreement with those of Land and McGovern (4) who reported that pretreating lambs with progesterone before induced ovulation resulted
196
DECEMBER
1975
VOL. 4 NO. 6
_-___
Ova recovered, %d
Ova fertilized, %
Per lamb.
% ova fertilized =
ova-..recovered _- _*-7 No. ova fertlllzed
No. ---
2.2 t 1.1 11.96 t 2.9" 3.28 t 1.1"
8.3 t 6.4 3.31t- 1.3 1.34 t 0.7 0.96 t 0.4 20.90 t 2.3
4.6 t 3.2 7.76 t 2.7 2.38 __ t 0.9 2.89 t 1.3 26.60 t 3.7
-_
16.6 t 11.4
35.3 t 14.6
37.7 t 14.1
30.90 t 3.6*
5.11 t 1.6"
13.2 t 4.9*
20
3.4 t 1.7
20
PHSG (i.u.1 1000 500
9.1t- 3.9
20
Mean significpntlv (PL.OS) greater than in lambs receivinp 500 i. u. PMSG. ** Mean sipnificantly (P<.Ol) less than in treated VOUIIS.
d
"Mean t _- standard error.
b
aFour SC1 of 25 mg at 3-dav intervals.
25.40 __ t 3.3
3.18 .._ t 1.4
2.24 t 1.1
x 100
8.40 t __0.4
..____--_.------__-
Kenroductive tract wt., pmb
----___
0.25 t 0.00
---
0.86 + O.l** 7.51 _+ 2.6
Follicular fluid wt., gmb
Coroora lutea wt.. gmb
Ovarian wt., gmb
4.0 + 2.0
20.9 + 11.5
7.5 t 3.5c
__-
lambs
NO. corpora luteab
No.
20
Control
PMSG (500 t 1000-_i.u.1 _~ Progesteronea t
Table I. ResDonse of 90-dav-old Ewe Lambs to Gonadotropic Hormone and-progesterone Treatment for Induction of 0~kation
10
Item --____-
--
THERIOGENOLOGY
in higher fertilizationrates. There was no significantdifference in weight of the ovaries, corpora lutea, follicularfluid or the reproductivetract due to progesteronetreatment. Average ovulation rate in lambs receiving 500 i.u. PMSG was 3.4 compared with an average of 13.2 from lambs injected with 1000 i.u. PMSG. The higher level of PMSG resulted in a significant(PL.05) increase in ovulation rate. Ovulation response was quite variable within gonadotropintreatment groups, ranging from 1 to 24 ovulations per lamb on the lower dosage and from 1 to 46 ovulationsat the higher level. Although the ovulation rate was higher in lambs receiving 1000 i.u. PMSG both percent ova recoveredand percent ova fertilized were lower in these lambs. The data show that ova produced by the 90-day-oldlamb are capable of fertilization,however, the actual percent fertilizedmay be very low. The low fertilizationrate may have resulted from an unfortuitousselectionof time of insemination relative to HCG injection. None of the lambs exhibitedestrus when checked daily after HCG injection. Ovaries were significantly(PC.05)heavier (11.96 vs. 3.31 gm) in lambs receiving 1000 i.u. PMSG. Similarly,corpora lutea, follicular fluid and reproductivetracts were significantly(PL.05) heavier from lambs receiving the higher PMSG dosage. A comparisonof the two levels of PMSG with or without pretreatment with progesteroneis shown in Table II. Although percent ova recovered was higher in the progesterone-pretreated ewes at the 500 i.u. PMSG dose (51.2%) compared to the nonpretreatedlambs (11.1%) this differencewas nonsignificant. Within each PMSG treatment group there were no significantdifferencesbetween the progesteroneand nonprogesterone-pretreated ewes for any of the traits studied. Also, the PMSG x progesteroneinteractionswere nonsignificantfor all traits studied. A disturbingfact of this experimentwas the low ova recovery rate, the reason for which is not readily apparent. However, close examinationof the reproductivetracts may offer a possible explanation for failure to recover many ova. The large increase in the size of the ovary followinghormonal stimulationis illustratedby comparingthe size of the reproductivetracts of a control and a hormone-treatedlamb (Plate I, Figure 1). The photograph (Plate I, Figure 2) shows that increase in size of the infundibulumwas not proportionalto that for the stimulatedovary. It was thus physically impossiblefor the infundibulumto completelysurround the ovary and many of the ova would be lost in the body cavity.
198
DECEMBER 1975
VOL. 4 NO. 6
Item
z
Q\
.
2
d
ow
fertilized, % = ?!_%_?v?~~~ No. ova recovered
'Mean -_ t standard error.
bPer lamb.
aFour SC1 of 25 mp:at 3-day intervals.
100
18.99 t 1.37 __
Reproductive tract wt., gmb
x
0.64 + 0.17 -
Follicular fluid wt., an@
-__
11.1 + 13.0
2.7 + 0.75c -
10
0.94 t 0.21
--~----_._____~_I_~__--___---
PMSG (500 i.u.1
22.90 z, 2.86
1.27 t 0.58 -
1.74 t 0.95
31.80 __ + 3.45
5.71 ._ t 1.73
3.54 + 1.18
12.59 + 2.91
4.6 t 1.7
10.0 + 13.1 _4.20 t 1.76 -
17.9 f 10.2
12.3 + 4.4 _-
10
(1000 w---Fi.u.)
51.2 + 15.7 ._
4.1 t 2.3 -
10
t nrogesteronea PMSG..--(500 __.__. .__.i___. _-___. _ __---_-.____PMSG _.-.._ _--. i.u.1
Coroora lutea wt., mb
..^__..
30.20 + 3.98
4.51t- 1.62
3.02 t 0.79
11.32 t 2.99
__-
15.6 t 12.9
14.1t- 4.6
10
PMSG (1000 i.u.1 t ---progesterone
Fesnonnssof SO-dav-old Ewe Lambs to Gonadotronic Hormones with or without Pretreatment with Progesterone
2.42 t 0.43 -
__.._
--P
Table II.
Ovarian wt., gnb
Ova fertilized, td
Ova recovered, "6
;io . cornora luteab
iJo. lambs
_-_.__-
m
Et
B
3
THERIOGENOLOGY PLATE I
200
Figure 1.
Reproductive tract of untreated lamb on left and treated on right. Note relative size of ovaries.
Figure 2.
Close-up of stimulated ovary and extended fimbria of oviduct. The fimbria is too small to engulf the stimulated ovary and consequently many ovulated ova drop into the body cavity and cannot be recovered.
DECEMBER 1975
VOL. 4 NO. 6
THERIOGENOLOGY
REFFIRFINCES 1.
Black, W. G., Ulberg, L. C., Christian, R. E. and Casida, L. E. Ovulation and Fertilization in the Hormone-stimulated Calf. J. Dairy Sci. -36~274 (1953).
2.
Casida, L. E., Meyer, R. K., McShan, W. H. and Wisnicky, W. Effects of Pituitary Gonadotrcphins on the Ovaries and the Induction of Superfecundity in Cattle. Am. J. Vet. Res. %:76 (1943).
3.
Onuma, H., Hahn, J. and Foote, R. H. Factors Affecting Superovulation, Fertilization and Recovery of Superovulated Ova in Prepuberal Cattle. J. Reprod. Fert. =:119 (1970).
4.
Land, R. B. and McGovern, P. T. Ovulation and Fertilization in the Lamb. J. Reprod. Fert. 15:325 (19681.
5.
Mansour, A. M. The Hormonal Control of Ovulation in the Immature Lamb. J. Agric. Sci. =:87 (1959).
DECEMBER 1975
VOL. 4 NO. 6
201
INDUCTION OF OVULATION AND FERTILIZATION IN THE 90-DAY-OLD EWE LAMBta) T. P. Goerkefb) and R. H. Dutt Department of Animal Sciences, University of Kentucky Lexington, Kentucky 40506 (Received for publication:
October 20, 1975)
ABSTRACT The induction of ovulation and fertilization was studied in 50 sexually immature crossbred ewe lambs which received 500 or 1000 i.u. PMSG with or without pretreatment with progesterone. Pretreatment with progesterone did not significantly affect ovulation, fertilization or ova recovery rates. Also, progesterone pretreatment did not significantly affect weight of the ovaries, corpora lutea, follicular fluid or the reproductive tract. Lambs receiving 1000 i.u. PMSG had a significantly (PC.05) higher ovulation rate (13.2) than lambs receiving 500 i.u. PMSG (3.4). Weights of the ovaries, corpora lutea, follicular fluid and the reproductive tract were significantly (P1.05) higher in ewes receiving 1000 i.u. PMSG. None of the lambs exhibited estrus when checked daily following HCG injection. The low ova recovery rate (16 to 38 percent) was postulated to be due to failure of the fimbria of the infundibulum to surround the enlarged stimulated ovary and pick up released ova. INTRODUCTION Induction of ovulation, fertilization and successful ova recovery in the sexually immature female , if followed by ova transfer techniques, could result in shortening the generation interval and thereby enhance genetic improvement of livestock. Ovulation has been induced in the calf (1,2,3) and in the sexually immature ewe lamb (4,5). However, information is scanty on fertility and on the recovery rates of ova in the immature sheep. The present study was initiated to determine ovulation, fertilization and ova recovery rates in sexually immature ewe lambs following hormonal induction of ovulation.
(a) Published with approval of the Director of the University of Kentucky Agricultural Experiment Station as Journal Article 74-s-139. (b)Present address: Department of Animal and Food Sciences, University of Wisconsin, River Falls, Wisconsin 54022.
DECEMBER 1975
VOL. 4 NO. 6
195
THERIOGENOLOGY MATERIALS AND METHODS -Fifty sexually immature crossbred (Southdown x Western) ewe lambs, averaging 90 days of age (range 83 to 103 days) and 24 kg in weight, were randomly allotted on July 1 to 5 treatment croups of 10 lambs each. Prior to treatment lambs were maintained on a diet of high quality alfalfa hay and shelled corn. Ovulation was induced by subcutaneous injection (SC11 of Pregnant Mare Serum Gonadotropin (PMSG) at two levels (500 or 1000 i.u.) followed 2 days later with an intramuscular injection of 500 i.u. of Human Chorionic Gonadotropin (HCG). The effect of pretreatment with 25 mp of progesterone (four SC1 at d-day intervals) was also studied. Ten ewe lambs were slaughtered for control data and, in a factorial design, the remaininp 40 lambs were used to determine the effects of progesterone pretreatment at two levels of PMSG. All lambs were artificially inseminated on the day of, and one day following HCG injection, with 1 ml of freshly collected ram semen diluted 1 to 10 with previously heated whole milk. Lambs were checked for estrus daily with aproned teaser rams following HCG injection. The lambs were slaughtered 5 days after HCG injection. Immediately following slaughter the reproductive tracts were removed from the carcass and the number of corpora lutea counted to estimate ovulation rate. Ova were recovered from the oviduct and uterus by flushing the contents into a watch glass with 0.85% saline. Following recovery, ova were stained and examined at 430X for evidence of cleavage and the presence of spermatozoa in the zona pellucida. After ovaries were dissected free and weighed, the corpora lutea were dissected from the ovaries and weighed. The remaining portion of the ovaries were cut into l-mm slices which were blotted free of fluid with a paper towel. The slices were then weighed and the follicular fluid weipht determined by difference. RESULTS AND DISCUSSION Ovaries from control lambs were immature, with an average weight varying from 7 to 26% of that of ovaries from treated lambs (Table I). None of the lambs in the control group ovulated whereas all treated lambs did. The mean ovulation rate (9.1) in progesterone-pretreated lambs was higher but not sipnificantly different from the ovulation rate (7.5) in lambs not pretreated with progesterone. Percent ova recovered and percent ova fertilized were 37.7 and 4.6, respectively, for the progesterone-pretreated lambs and 20.9 and 4.0, respectively, for the non-pretreated lambs. Although both ova recovery and percent ova fertilized were slightly higher in the progesterone-pretreated lambs, differences due to progesterone treatment were not statistically significant. At the onset of the study it was postulated that progesterone might improve the uterine environment and result in higher fertility. However, this was not the case. These data are not in agreement with those of Land and McGovern (4) who reported that pretreating lambs with progesterone before induced ovulation resulted
196
DECEMBER
1975
VOL. 4 NO. 6
_-___
Ova recovered, %d
Ova fertilized, %
Per lamb.
% ova fertilized =
ova-..recovered _- _*-7 No. ova fertlllzed
No. ---
2.2 t 1.1 11.96 t 2.9" 3.28 t 1.1"
8.3 t 6.4 3.31t- 1.3 1.34 t 0.7 0.96 t 0.4 20.90 t 2.3
4.6 t 3.2 7.76 t 2.7 2.38 __ t 0.9 2.89 t 1.3 26.60 t 3.7
-_
16.6 t 11.4
35.3 t 14.6
37.7 t 14.1
30.90 t 3.6*
5.11 t 1.6"
13.2 t 4.9*
20
3.4 t 1.7
20
PHSG (i.u.1 1000 500
9.1t- 3.9
20
Mean significpntlv (PL.OS) greater than in lambs receivinp 500 i. u. PMSG. ** Mean sipnificantly (P<.Ol) less than in treated VOUIIS.
d
"Mean t _- standard error.
b
aFour SC1 of 25 mg at 3-dav intervals.
25.40 __ t 3.3
3.18 .._ t 1.4
2.24 t 1.1
x 100
8.40 t __0.4
..____--_.------__-
Kenroductive tract wt., pmb
----___
0.25 t 0.00
---
0.86 + O.l** 7.51 _+ 2.6
Follicular fluid wt., gmb
Coroora lutea wt.. gmb
Ovarian wt., gmb
4.0 + 2.0
20.9 + 11.5
7.5 t 3.5c
__-
lambs
NO. corpora luteab
No.
20
Control
PMSG (500 t 1000-_i.u.1 _~ Progesteronea t
Table I. ResDonse of 90-dav-old Ewe Lambs to Gonadotropic Hormone and-progesterone Treatment for Induction of 0~kation
10
Item --____-
--
THERIOGENOLOGY
in higher fertilizationrates. There was no significantdifference in weight of the ovaries, corpora lutea, follicularfluid or the reproductivetract due to progesteronetreatment. Average ovulation rate in lambs receiving 500 i.u. PMSG was 3.4 compared with an average of 13.2 from lambs injected with 1000 i.u. PMSG. The higher level of PMSG resulted in a significant(PL.05) increase in ovulation rate. Ovulation response was quite variable within gonadotropintreatment groups, ranging from 1 to 24 ovulations per lamb on the lower dosage and from 1 to 46 ovulationsat the higher level. Although the ovulation rate was higher in lambs receiving 1000 i.u. PMSG both percent ova recoveredand percent ova fertilized were lower in these lambs. The data show that ova produced by the 90-day-oldlamb are capable of fertilization,however, the actual percent fertilizedmay be very low. The low fertilizationrate may have resulted from an unfortuitousselectionof time of insemination relative to HCG injection. None of the lambs exhibitedestrus when checked daily after HCG injection. Ovaries were significantly(PC.05)heavier (11.96 vs. 3.31 gm) in lambs receiving 1000 i.u. PMSG. Similarly,corpora lutea, follicular fluid and reproductivetracts were significantly(PL.05) heavier from lambs receiving the higher PMSG dosage. A comparisonof the two levels of PMSG with or without pretreatment with progesteroneis shown in Table II. Although percent ova recovered was higher in the progesterone-pretreated ewes at the 500 i.u. PMSG dose (51.2%) compared to the nonpretreatedlambs (11.1%) this differencewas nonsignificant. Within each PMSG treatment group there were no significantdifferencesbetween the progesteroneand nonprogesterone-pretreated ewes for any of the traits studied. Also, the PMSG x progesteroneinteractionswere nonsignificantfor all traits studied. A disturbingfact of this experimentwas the low ova recovery rate, the reason for which is not readily apparent. However, close examinationof the reproductivetracts may offer a possible explanation for failure to recover many ova. The large increase in the size of the ovary followinghormonal stimulationis illustratedby comparingthe size of the reproductivetracts of a control and a hormone-treatedlamb (Plate I, Figure 1). The photograph (Plate I, Figure 2) shows that increase in size of the infundibulumwas not proportionalto that for the stimulatedovary. It was thus physically impossiblefor the infundibulumto completelysurround the ovary and many of the ova would be lost in the body cavity.
198
DECEMBER 1975
VOL. 4 NO. 6
Item
z
Q\
.
2
d
ow
fertilized, % = ?!_%_?v?~~~ No. ova recovered
'Mean -_ t standard error.
bPer lamb.
aFour SC1 of 25 mp:at 3-day intervals.
100
18.99 t 1.37 __
Reproductive tract wt., gmb
x
0.64 + 0.17 -
Follicular fluid wt., an@
-__
11.1 + 13.0
2.7 + 0.75c -
10
0.94 t 0.21
--~----_._____~_I_~__--___---
PMSG (500 i.u.1
22.90 z, 2.86
1.27 t 0.58 -
1.74 t 0.95
31.80 __ + 3.45
5.71 ._ t 1.73
3.54 + 1.18
12.59 + 2.91
4.6 t 1.7
10.0 + 13.1 _4.20 t 1.76 -
17.9 f 10.2
12.3 + 4.4 _-
10
(1000 w---Fi.u.)
51.2 + 15.7 ._
4.1 t 2.3 -
10
t nrogesteronea PMSG..--(500 __.__. .__.i___. _-___. _ __---_-.____PMSG _.-.._ _--. i.u.1
Coroora lutea wt., mb
..^__..
30.20 + 3.98
4.51t- 1.62
3.02 t 0.79
11.32 t 2.99
__-
15.6 t 12.9
14.1t- 4.6
10
PMSG (1000 i.u.1 t ---progesterone
Fesnonnssof SO-dav-old Ewe Lambs to Gonadotronic Hormones with or without Pretreatment with Progesterone
2.42 t 0.43 -
__.._
--P
Table II.
Ovarian wt., gnb
Ova fertilized, td
Ova recovered, "6
;io . cornora luteab
iJo. lambs
_-_.__-
m
Et
B
3
THERIOGENOLOGY PLATE I
200
Figure 1.
Reproductive tract of untreated lamb on left and treated on right. Note relative size of ovaries.
Figure 2.
Close-up of stimulated ovary and extended fimbria of oviduct. The fimbria is too small to engulf the stimulated ovary and consequently many ovulated ova drop into the body cavity and cannot be recovered.
DECEMBER 1975
VOL. 4 NO. 6
THERIOGENOLOGY
REFFIRFINCES 1.
Black, W. G., Ulberg, L. C., Christian, R. E. and Casida, L. E. Ovulation and Fertilization in the Hormone-stimulated Calf. J. Dairy Sci. -36~274 (1953).
2.
Casida, L. E., Meyer, R. K., McShan, W. H. and Wisnicky, W. Effects of Pituitary Gonadotrcphins on the Ovaries and the Induction of Superfecundity in Cattle. Am. J. Vet. Res. %:76 (1943).
3.
Onuma, H., Hahn, J. and Foote, R. H. Factors Affecting Superovulation, Fertilization and Recovery of Superovulated Ova in Prepuberal Cattle. J. Reprod. Fert. =:119 (1970).
4.
Land, R. B. and McGovern, P. T. Ovulation and Fertilization in the Lamb. J. Reprod. Fert. 15:325 (19681.
5.
Mansour, A. M. The Hormonal Control of Ovulation in the Immature Lamb. J. Agric. Sci. =:87 (1959).
DECEMBER 1975
VOL. 4 NO. 6
201