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Ovum recovery in superovulated cows and cleavage rates in the fertilized ova
THERIOGENOLOGY
OVUM RECOVERY IN SUPEROVULATED COWS AND CLEAVAGE RATES IN THE FERTILIZED OVA J. W. McGaught D. Olds and D. D. Kratzer Department of Animal Sciences University of Kentucky Lexington, Kentucky 40506 Received for publication July 29, 1974.
SUMMARY Four groups of 10 cows were given one, two, three, or four thousand i.u. of PMS on Day 16 of the estrous cycle. On Day 19, the cows were injected with lo-15 mg of estrogen. At the ensuing estrus, the cows were bred and given 2,000 i.u. of HCG. The cows were slaughtered for ovum recovery 3-7 days after breeding. On the average, the cows produced 10.3 + 1.6 ovulations with a range from 0 to 41. Each 1,000 i.u. of PMS was associated with an increase of 5.83 + 1.18 in number of ovulations. The variation among cows within treatment groups was not attributable to differences in age or body weight. On the average, 51.5% of the ovulated ova were recovered, with the recovery rate being higher (64.2%) for ova in the oviducts and lower (34.21%)for those in the uterus. Ovarian length at the time of slaughter was directly related to PMS dosage and a smaller proportion of the ova were recovered from animals having largest ovaries. Only 38.45,of 211 recovered ova were fertilized. It was estimated that cleavages occurred at intervals of 12.6 f. 23.6 hours. INTRODUCTION There has been considerable interest in the superovulation of cattle for inducing twins, the production of larger numbers of offspring from outstanding females, the study of ova, and perhaps for obtaining a more quantitative measure of fertilization rates. Foote and Cnuma (2) summarized data from six studies indicating that 55% of the ovulated wa were recovered and 63% of these were fertilized. While hormones used and treatment schedules have been variable, the most useful methods would seem to be based upon pregnant mare serum (PMS) given on Day 16, estradiol benzoate on Day 19 or 20 and human chorionic gonadotropin (HCG) on the day of estrus as reported by Scanlon (6), or upon repeated doses of purified follicle stimulating hormone (FSH) as reported by Bellows et al. (1). Scanlon (7) has recently reported that the administration of 3,000 i-u. of PMS on Day
'The investigation reported in this paper (No. 71-5-117) is in connection with a project of the Kentuckv Agricultural Experiment Station and is published with approval of the director. 2 Present address: 811 E. Georgia St., Shawnee, Okla. 74801.
JUNK 1974
VOL. 1 NO. 6
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THERIOGENOLOGY
16 resulted in 81.6% of 152 cows being superovulated with an average of 12.1 ovulations. He also reported that ova were recovered from 62.1% of the ovulations (8) and that 54.1% of the recovered ova were fertilized (9). Turman et al. (10) injected 1,500 i.u. of PMS between Days 3 and 6 and 2,000 i.u. between Days 16 and 18 of the cycle, followed by 2,500 i.u. of HCG on the day of estrus. Among 81 cows so treated, 64.2% conceived when bred and produced 29 single births, 12 sets of twins, 8 triplets, 2 quadruplets, and 1 set of quintuplets. Henricks s a. (4) injected a group of 8 cows with 1,600 i.u. PMS on Day 16 and obtained 6.4 + 5.0 ovulations per cow while doses of 3,200 i.u. produced 12.6 + 4.7 ovulations. They recovered 64.2% of the ova and 75.4% were fertilized. The present study was conducted to determine the ovarian response to graded doses of PMS and to estimate the recovery rates, fertilization rates and cleavage rates of superovulated ova. MATERIALS AND METHODS A total of 38 Holstein and 2 Jersey females ranging from 19 months to 10 years of age were fed on good quality grass pasture or alfalfa hay and corn silage. The animals were either non-lactating or were prematurely dried off wlnen placed on the experiment. The animals were observed twice daily for signs of estrus and as they came into estrus, they were assigned to receive 1,000, 2,000, 3,000 or 4,000 i.u. of PMS (Ayerst) intramuscularly on Day 16 of the cycle (Day 0 = day of estrus). All of the cows were given lo-15 mg of estradiol benzoate or diethylstilbestrol subcutaneously on Day 19 of the cycle. When estrus occurred, the cows were given 2,000 i.u. of HCG (Ayerst) intravenously. Most were bred by natural service and the remainder by artificial insemination using frozen semen. The cows were slaughtered 3-7 days after estrus. The oviducts were separated from the uterus and were flushed from the fimbriated end with 5 ml of saline distributed into 4 watch glasses. If sufficient ova were not found to account for all of the ovulation points, the oviducts were flushed again from the opposite direction; the uterine horns were flushed with about 40 ml of saline which was poured into a separatory funnel for examination of successive samples drawn from the bottom. RESULTS AND DISCUSSION PMS dose had a highly significant linear effect on the number of ovulations. The regression coefficient indicated that each additional 1,000 i.u. of PMS was associated with 5.83 + 1.18 more ovulations. The variation among cows within treatment groups was considerable, as shown by the standard errors in Table 1. The range for all cows was 0 to 41 ovulations. The differences among cows were found not to be caused by differences in age or body weight.
214
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VOL. 1 NO. 6
THERIOGENOLOGY
TABLE1 MEANS AND STANDARD ERRORS FOR NUMBERS OF OVULATIONS, OVA RECOVERED, FERTILIZED OVA RECOVERED AND OVARIAN LENGTHS FOR COWS GIVEN VARYING DOSES OF PMS ON DAY 16 OF AN ESTROUS CYCLE (N = 10).
Ovulations Mean * S.E.
Treatment 1,OOCli.u. 2,00C i.u. 3,00C i.u. 4,00C i.u.
PMS PMS PMS PMS
2.2 6.5 12.8 19.5
0.6 2.0 2.8 3.7
Numbers of Ova Recovered Fertilized Mean? S.E. Mean * S.E. 1.3 3.9 6.6 9.3
0.4 1.5 2.0 2.2
0.6 0.3 4.2 3.1
0.3 0.2 1.8 1.1
Ovarian Length (cm) Meant S.E. 3.5 4.2 5.8 6.2
0.08 0.28 0.54 0.55
Ihe 40 cows were slaughtered 3-7 days following estrus and on the average, 51.5% of the ovulations were represented by recovered ova. Of 211 ova recovered, 97 were from cows in which ova were found only in the oviducts (at 4.50 +_ 0.29 days after the administration of HCG during estrus). Since these cows had 151 discernible ovulation points, 64.2% of the ovulated ova were recovered. On the other hand, there were 41 ova recovered from cows in which ova were found only in the uterus (at 5.72 + 0.48 days). In these cows, only 34.2% of the ovulated ova were recovered. The remaining 73 ova were recovered from cows in which some ova were found in the oviducts and some in the uterus; 52.5% of the ovulated ova were recovered. shrariansize increased linearly with increasing doses of PMS. The a*rerageweight of both ovaries was 18.6 gm for cows receiving 1,000 i.u. of PMS, 34.7 gm for those receiving 2,000, 99.3 gm for 3,000 and 116.2 gm for 4,000. Ovarian length was more highly correlated with number of ovulations (r = 0.7) than was thickness, width, or weight of the ovaries. The partial correlation coefficient between ovarian length and total ova recovered, independent of numbe:: of ovulation points and time from HCG injection to slaughter, was -.46 indicating that large ovaries were associated with smaller percentages of ova recovered. Each 1,000 i.u. of PMS was associated with an increase of 1.0 + 0.08 cm in ovarian length. In this study, 38.4% of the 211 recovered ova were fertilized. It was not possible to explain the reason for such a low fertilization ::ate. While it may have been preferable to breed all cows artificially to a bull of known high fertility, it appeared that higher fertility was obtained by natural service, but the difference was not significant for the small numbers involved. Actually this exper:.mentwas not designed to test the differences in type of service and it is unfortunate that such differences were included. It is also perhaps possible that the administration of estrogen on Day 19 had some undesired effect on the results. However, the rather large variability among cows in ovarian response to PMS as
JUNE, 1974 VOL. 1 NO. 6
215
THERIOGENOLOGY
obtained in this study, was not greatly different from that obtained by others (4). The 81 fertilized ova of this study were recovered from 20 cows which were slaughtered 69-168 hours after the administration of HCG during estrus. For each cow, the average number of blastomeres per ovum was converted to log2, thus indicating the number of cleavages. The regression coefficient between number of cleavages and length of time from HCG injection to slaughter was found to be linear and indicated that cleavages occurred every 12.6 + 23.6 hours. For comparison, similar techniques were applied to the data of Hamilton and Laing (3) for 34 singly produced ova. Their data indicated that cleavages occurred at intervals of 14.1 +_ 17.6 hours. The difference between these cleavage rates was not statistically significant. However, it is apparent that a high degree of variability exists among ova and cows with regard to cleavage rates. Pooling the above data with that of Olds et al. (5) indicated that 2, 4, 8 and 16-cell ova can be expected at 2.54, 3.18, 3.81 and 4.44 days, respectively, after the time of service or insemination, which in this experiment also coincided with the injection of HCG. These estimates may be of some value in selecting times for ovum recovery in ovum transfer operations. REFERENCES
216
1.
Bellows, R. A., Anderson, D. C. and Short, R. E. Dose-Response Relationships in Synchronized Beef Heifers Treated with Follicle Stimulating Hormone. J. Animal Sci. 28:638 (1969).
2.
Foote, R. H. and Onuma, H. Superovulation, Ovum Collection, Culture and Transfer. A Review. J. Dairy Sci. 53:1681 (1970).
3.
Hamilton, W. S. and Laing, S. A. Development of the Egg of the Cow Up to the Stage of Blastocyst Formation. J. Anat. 8:194 (1946).
4.
Henricks, D. M., Hill, J. R. Jr., Dickey, J. F. and Lamond, D. R. Plasma Hormone Levels in Beef Cows with Induced Multiple Ovulations. J. Reprod. Fert. 35:225 (1973).
5.
Olds, D., Graden, A. P., Mochow, C. R. and Mutter, L. R. Observations on the Size of Bovine Ova. J. Dairy Sci. 51:215 (1968).
6.
Scanlon, P. F. Hormonal Induction of Multiple Ovulations in Cattle. VIth Intern. Cong. Animal Reprod. Art. Insem. (Paris) 1:801 (1968).
7.
Scanlon, P. F. Ovarian Response of Cattle to 3,000 IU PMSG. (Abstr.) J. Animal Sci. 35:253 (1972).
8.
Scanlon, P. F. Recovering of Eggs from Superovulated Cattle. (Abstr.) J. Animal Sci. 35:253 (1972).
JUNE
1974
VOL.
1 NO. 6
THERIOGENOLOGY
9.
10.
Scanlon, P. F. Cleavage Rates of Eggs from Superovulated Cattle, (Abstr.) J. Animal Sci. 35:253 (1972). Tut-man,E. J., Laster, D. B., Renbarger, R. E. and Stephens, D. F. Multiple Bikhs in Beef Cows Treated with Equine Gonadotropin (PMS) and Chorionic Gonadotropin (KG). J. Animal Sci. 32:962 (1971).
JUNE
1974
VOL. 1 NO. 6
217
OVUM RECOVERY IN SUPEROVULATED COWS AND CLEAVAGE RATES IN THE FERTILIZED OVA J. W. McGaught D. Olds and D. D. Kratzer Department of Animal Sciences University of Kentucky Lexington, Kentucky 40506 Received for publication July 29, 1974.
SUMMARY Four groups of 10 cows were given one, two, three, or four thousand i.u. of PMS on Day 16 of the estrous cycle. On Day 19, the cows were injected with lo-15 mg of estrogen. At the ensuing estrus, the cows were bred and given 2,000 i.u. of HCG. The cows were slaughtered for ovum recovery 3-7 days after breeding. On the average, the cows produced 10.3 + 1.6 ovulations with a range from 0 to 41. Each 1,000 i.u. of PMS was associated with an increase of 5.83 + 1.18 in number of ovulations. The variation among cows within treatment groups was not attributable to differences in age or body weight. On the average, 51.5% of the ovulated ova were recovered, with the recovery rate being higher (64.2%) for ova in the oviducts and lower (34.21%)for those in the uterus. Ovarian length at the time of slaughter was directly related to PMS dosage and a smaller proportion of the ova were recovered from animals having largest ovaries. Only 38.45,of 211 recovered ova were fertilized. It was estimated that cleavages occurred at intervals of 12.6 f. 23.6 hours. INTRODUCTION There has been considerable interest in the superovulation of cattle for inducing twins, the production of larger numbers of offspring from outstanding females, the study of ova, and perhaps for obtaining a more quantitative measure of fertilization rates. Foote and Cnuma (2) summarized data from six studies indicating that 55% of the ovulated wa were recovered and 63% of these were fertilized. While hormones used and treatment schedules have been variable, the most useful methods would seem to be based upon pregnant mare serum (PMS) given on Day 16, estradiol benzoate on Day 19 or 20 and human chorionic gonadotropin (HCG) on the day of estrus as reported by Scanlon (6), or upon repeated doses of purified follicle stimulating hormone (FSH) as reported by Bellows et al. (1). Scanlon (7) has recently reported that the administration of 3,000 i-u. of PMS on Day
'The investigation reported in this paper (No. 71-5-117) is in connection with a project of the Kentuckv Agricultural Experiment Station and is published with approval of the director. 2 Present address: 811 E. Georgia St., Shawnee, Okla. 74801.
JUNK 1974
VOL. 1 NO. 6
213
THERIOGENOLOGY
16 resulted in 81.6% of 152 cows being superovulated with an average of 12.1 ovulations. He also reported that ova were recovered from 62.1% of the ovulations (8) and that 54.1% of the recovered ova were fertilized (9). Turman et al. (10) injected 1,500 i.u. of PMS between Days 3 and 6 and 2,000 i.u. between Days 16 and 18 of the cycle, followed by 2,500 i.u. of HCG on the day of estrus. Among 81 cows so treated, 64.2% conceived when bred and produced 29 single births, 12 sets of twins, 8 triplets, 2 quadruplets, and 1 set of quintuplets. Henricks s a. (4) injected a group of 8 cows with 1,600 i.u. PMS on Day 16 and obtained 6.4 + 5.0 ovulations per cow while doses of 3,200 i.u. produced 12.6 + 4.7 ovulations. They recovered 64.2% of the ova and 75.4% were fertilized. The present study was conducted to determine the ovarian response to graded doses of PMS and to estimate the recovery rates, fertilization rates and cleavage rates of superovulated ova. MATERIALS AND METHODS A total of 38 Holstein and 2 Jersey females ranging from 19 months to 10 years of age were fed on good quality grass pasture or alfalfa hay and corn silage. The animals were either non-lactating or were prematurely dried off wlnen placed on the experiment. The animals were observed twice daily for signs of estrus and as they came into estrus, they were assigned to receive 1,000, 2,000, 3,000 or 4,000 i.u. of PMS (Ayerst) intramuscularly on Day 16 of the cycle (Day 0 = day of estrus). All of the cows were given lo-15 mg of estradiol benzoate or diethylstilbestrol subcutaneously on Day 19 of the cycle. When estrus occurred, the cows were given 2,000 i.u. of HCG (Ayerst) intravenously. Most were bred by natural service and the remainder by artificial insemination using frozen semen. The cows were slaughtered 3-7 days after estrus. The oviducts were separated from the uterus and were flushed from the fimbriated end with 5 ml of saline distributed into 4 watch glasses. If sufficient ova were not found to account for all of the ovulation points, the oviducts were flushed again from the opposite direction; the uterine horns were flushed with about 40 ml of saline which was poured into a separatory funnel for examination of successive samples drawn from the bottom. RESULTS AND DISCUSSION PMS dose had a highly significant linear effect on the number of ovulations. The regression coefficient indicated that each additional 1,000 i.u. of PMS was associated with 5.83 + 1.18 more ovulations. The variation among cows within treatment groups was considerable, as shown by the standard errors in Table 1. The range for all cows was 0 to 41 ovulations. The differences among cows were found not to be caused by differences in age or body weight.
214
JUNE 1974
VOL. 1 NO. 6
THERIOGENOLOGY
TABLE1 MEANS AND STANDARD ERRORS FOR NUMBERS OF OVULATIONS, OVA RECOVERED, FERTILIZED OVA RECOVERED AND OVARIAN LENGTHS FOR COWS GIVEN VARYING DOSES OF PMS ON DAY 16 OF AN ESTROUS CYCLE (N = 10).
Ovulations Mean * S.E.
Treatment 1,OOCli.u. 2,00C i.u. 3,00C i.u. 4,00C i.u.
PMS PMS PMS PMS
2.2 6.5 12.8 19.5
0.6 2.0 2.8 3.7
Numbers of Ova Recovered Fertilized Mean? S.E. Mean * S.E. 1.3 3.9 6.6 9.3
0.4 1.5 2.0 2.2
0.6 0.3 4.2 3.1
0.3 0.2 1.8 1.1
Ovarian Length (cm) Meant S.E. 3.5 4.2 5.8 6.2
0.08 0.28 0.54 0.55
Ihe 40 cows were slaughtered 3-7 days following estrus and on the average, 51.5% of the ovulations were represented by recovered ova. Of 211 ova recovered, 97 were from cows in which ova were found only in the oviducts (at 4.50 +_ 0.29 days after the administration of HCG during estrus). Since these cows had 151 discernible ovulation points, 64.2% of the ovulated ova were recovered. On the other hand, there were 41 ova recovered from cows in which ova were found only in the uterus (at 5.72 + 0.48 days). In these cows, only 34.2% of the ovulated ova were recovered. The remaining 73 ova were recovered from cows in which some ova were found in the oviducts and some in the uterus; 52.5% of the ovulated ova were recovered. shrariansize increased linearly with increasing doses of PMS. The a*rerageweight of both ovaries was 18.6 gm for cows receiving 1,000 i.u. of PMS, 34.7 gm for those receiving 2,000, 99.3 gm for 3,000 and 116.2 gm for 4,000. Ovarian length was more highly correlated with number of ovulations (r = 0.7) than was thickness, width, or weight of the ovaries. The partial correlation coefficient between ovarian length and total ova recovered, independent of numbe:: of ovulation points and time from HCG injection to slaughter, was -.46 indicating that large ovaries were associated with smaller percentages of ova recovered. Each 1,000 i.u. of PMS was associated with an increase of 1.0 + 0.08 cm in ovarian length. In this study, 38.4% of the 211 recovered ova were fertilized. It was not possible to explain the reason for such a low fertilization ::ate. While it may have been preferable to breed all cows artificially to a bull of known high fertility, it appeared that higher fertility was obtained by natural service, but the difference was not significant for the small numbers involved. Actually this exper:.mentwas not designed to test the differences in type of service and it is unfortunate that such differences were included. It is also perhaps possible that the administration of estrogen on Day 19 had some undesired effect on the results. However, the rather large variability among cows in ovarian response to PMS as
JUNE, 1974 VOL. 1 NO. 6
215
THERIOGENOLOGY
obtained in this study, was not greatly different from that obtained by others (4). The 81 fertilized ova of this study were recovered from 20 cows which were slaughtered 69-168 hours after the administration of HCG during estrus. For each cow, the average number of blastomeres per ovum was converted to log2, thus indicating the number of cleavages. The regression coefficient between number of cleavages and length of time from HCG injection to slaughter was found to be linear and indicated that cleavages occurred every 12.6 + 23.6 hours. For comparison, similar techniques were applied to the data of Hamilton and Laing (3) for 34 singly produced ova. Their data indicated that cleavages occurred at intervals of 14.1 +_ 17.6 hours. The difference between these cleavage rates was not statistically significant. However, it is apparent that a high degree of variability exists among ova and cows with regard to cleavage rates. Pooling the above data with that of Olds et al. (5) indicated that 2, 4, 8 and 16-cell ova can be expected at 2.54, 3.18, 3.81 and 4.44 days, respectively, after the time of service or insemination, which in this experiment also coincided with the injection of HCG. These estimates may be of some value in selecting times for ovum recovery in ovum transfer operations. REFERENCES
216
1.
Bellows, R. A., Anderson, D. C. and Short, R. E. Dose-Response Relationships in Synchronized Beef Heifers Treated with Follicle Stimulating Hormone. J. Animal Sci. 28:638 (1969).
2.
Foote, R. H. and Onuma, H. Superovulation, Ovum Collection, Culture and Transfer. A Review. J. Dairy Sci. 53:1681 (1970).
3.
Hamilton, W. S. and Laing, S. A. Development of the Egg of the Cow Up to the Stage of Blastocyst Formation. J. Anat. 8:194 (1946).
4.
Henricks, D. M., Hill, J. R. Jr., Dickey, J. F. and Lamond, D. R. Plasma Hormone Levels in Beef Cows with Induced Multiple Ovulations. J. Reprod. Fert. 35:225 (1973).
5.
Olds, D., Graden, A. P., Mochow, C. R. and Mutter, L. R. Observations on the Size of Bovine Ova. J. Dairy Sci. 51:215 (1968).
6.
Scanlon, P. F. Hormonal Induction of Multiple Ovulations in Cattle. VIth Intern. Cong. Animal Reprod. Art. Insem. (Paris) 1:801 (1968).
7.
Scanlon, P. F. Ovarian Response of Cattle to 3,000 IU PMSG. (Abstr.) J. Animal Sci. 35:253 (1972).
8.
Scanlon, P. F. Recovering of Eggs from Superovulated Cattle. (Abstr.) J. Animal Sci. 35:253 (1972).
JUNE
1974
VOL.
1 NO. 6
THERIOGENOLOGY
9.
10.
Scanlon, P. F. Cleavage Rates of Eggs from Superovulated Cattle, (Abstr.) J. Animal Sci. 35:253 (1972). Tut-man,E. J., Laster, D. B., Renbarger, R. E. and Stephens, D. F. Multiple Bikhs in Beef Cows Treated with Equine Gonadotropin (PMS) and Chorionic Gonadotropin (KG). J. Animal Sci. 32:962 (1971).
JUNE
1974
VOL. 1 NO. 6
217