ELSEVIER

COMPARISON OF SYNCHRONIZATION OF OVULATION AND INDUCTION OF ESTRUS AS THERAPEUTIC STRATEGIES FOR BOVINE OVARIAN CYSTS IN THE DAIRY COW

J.A. Bartolome,l L.F. Archbald, la P. MorresIey,’ J. Hemandez, ’ 5 Tran,’ D. Kelbert,2 K. Long,2 C. A. Risco, and W.W. Thatcher

1 Department of Large Animal Clinical Sciences, College of Veterinary Medicine

2 University of Florida, Gainesville, FL 32610

3 North Florida Holsteins (NFH), Inc., Bell, FL 32619 Department of Dairy & Poultry Sciences, University of Florida, Gainesville, FL 32610

Received for publication: July 8, 1999

Accepted: August 24, 1999

ABSTRACT

The benefit of using timed-insemination in lactating dairy cows for the treatment of ovarian cysts lies in the fact that cows do not have to be detected in estrus for insemination and achieving pregnancy. We compared the effectiveness of synchronization of ovulation with timed-insemination and induction of estrus with insemination at estrus in the treatment of bovine ovarian cysts in lactating dairy cows. After Day 65 postpartum, a total of 368 lactating dairy cows was divided into 3 groups. Cows in Group 1 (n= 209, normal, noncystic) were treated with 100 ug, im GnRH on Day 0; 25 mg, im PGF,, on Day 7; and 100 ug, im GnRH on Day 9 and then were time-inseminated 16 h later. Cows in Group 2 (n=76, abnormal, cystic) were treated with 100 ug, im G&I-I on Day 0; 25 mg, im PGF,, on Day 7; and 100 ug, im GnRH on Day 9 and time-inseminated 16 h later. Cows in Group 3 (n=83, abnormal, cystic ) were treated with 100 ug, im GnRH on Day 0; 25 mg, im PGF,, on Day 7; and inseminated at induced estrus within 7 d after treatment with PGF,,. Day 0 was the day of initiation of the study. Conception and pregnancy rates among groups were compared using logistic regression and adjusted for parity, time of year and days in milk. Conception and pregnancy rates of Group 1 cows (3 1.5%) were not significantly different horn those of Group 2 cows (23.6%). However, the pregnancy rate in normal cows (Group 1) was higher (P<O.Ol) than in cystic cows (Groups 2 and 3). Cows in Group 3 had a higher conception rate than cows in Group 2 (51.7%>23.6%; P<O.Ol). However, pregnancy rates for cows in Groups 2 (23.6%) and 3 (18%) were not significantly different. The finding indicated that synchronization of ovulation and timed- insemination resulted in pregnancy rates similar to those of synchronization of estrus and insemination at an induced estrus within 7 d for the treatment of ovarian cysts in lactating dairy cows. 0 2000 by Elsevier Science Inc.

Key words: dairy cows, ovarian cysts, GnRH, PGF,,

Acknowledgments This study was funded by the Florida Dairymen Milk Check-Off Program. We thank Don Bemrink for use of the herd and the entire staff of NFH, Inc., for assistance with this study.

aCorrespondence and reprint requests.

Theriogenology 53815-825, 2000 009%691X/00/$-see front matter 0 2000 by Elsevier Science Inc. PII SOO93-691 X(99)00276-9

816

INTRODUCTION

Theriogenology

Ovarian cysts are follicles which fail to ovulate at the time of estrus and occur spontaneously in a variety of mammalian species (9). They are an economic problem in the dairy cow because these cows are infertile as long as the condition persists (13). The rate of occurrence of this abnormal condition in dairy cows ranges from 6 to 30% (4,5,&l 3,18,26). Thus, a conservative estimate is that at least one million dairy cows are affected by ovarian cysts each year.

The exact cause of ovarian cysts is not presently known. However, some predisposing conditions include age, stress, high milk production and genetics. It has been suggested that an important component in the pathogenesis of this condition is the inappropriate, or lack of, release of hypothalamic gonadotrophin-releasing hormone (GnRH). Therefore, a therapeutic approach consists of the use of exogenous GnRH to release LH and thus potentially to effect either ovulation of an ovarian follicle or luteinization of the ovarian cyst. The resulting corpus luteum (CL) or luteinized ovarian cyst subsequently undergoes spontaneous regression, or can be lysed by exogenous prostaglandin F,, (PGFr,).

However, success with this therapeutic approach depends on the observation of estrus exhibited by these cows within 7 d after treatment with PGF,,. If estrus is not observed or exhibited, these cows cannot be inseminated. Therefore, detection of estrus is a major contributing factor to the success of this treatment approach (1). It would, therefore, seem advantageous if there were a scheme whereby cows could be successfully artificially inseminated without the need to detect estrus.

Recent research (6,7,16,20,21,23,27) has shown the success of a protocol using GnRH and PGF?, (OVSYNCH) to obtain pregnancy in lactating dairy cows without the need for detecting es&us. This protocol synchronizes ovulation within an S-h period from 24 to 32 h after the second injection of GnRH. Such precise synchrony allows for successful AI without the need for detecting estrus.

The hypothesis of this study was that this protocol could be used successfully as a therapeutic approach for bovine ovarian cysts in the lactating dairy cow. The objective of this study was to compare a protocol for synchronization of ovulation and timed-insemination with one involving synchronization of es&us and insemination at an induced estrus within 7 d in the treatment of bovine ovarian cysts in the lactating dairy cow.

MATERIALS AND METHODS

This study was conducted during the period of September 1997 to June 1998 in a large dairy herd (approximately 3000 milking cows) in north central Florida. Between 60 and 63 d post partum, all cows in the herd were subjected to treatment with bovine somatotropin (bST; 500 mg, id; Posilac, Monsanto Co., St. Louis, MO) every 14 d until milk production decreased to a minimum level established by the herd managers. Consequently, all cows were being treated with bST during the

Theriogenology 817

study period. The herd was on a reproductive herd health program and was visited weekly. All reproductive, health and management records were computerized using a cow-side computer program.

The milking herd (approximately 3000 cows) was divided into 8 different lots based on production criteria established by the managers of the herd. Cows from each of the lots were used during the period of study. The cows were milked 3 times a day and were kept in shaded lots between milking.

A total of 368 lactating dairy cows was used in this study. The cows were diagnosed as reproductively normal or abnormal (cystic) based on clinical findings obtained by palpation per rectum of the uterus and ovaries. Cows were considered normal if a CL was present on the ovaries and the uterus was completely involuted. Previously published criteria were used to determine presence of a CL (28) and an involuted uterus (25). Cows were considered cystic if there were no CL, and multiple follicles (>20 mm in diameter) were present on one or both ovaries in the absence of uterine tonicity (1,ll).

At the initiation of the study (Day 0), cows were divided randomly into 3 experimental groups. Cows in Group 1 (n=209; normal) were treated with GnRH (100 ug, im; Cystorelin, Rhone Merieux Inc., Athens, GA 30601) on Day 0, one luteolytic dosage of PGF,, (25 mg, im; Lutalyse, Upjohn Company, Kalamazoo, MI 49001) on Day 7, GnRH on Day 9, and then time-inseminated approximately 16 h after the second treatment with GnRH. Cows in Group 2 (n=76; abnormal, cystic) were treated with GnRH on Day 0, one luteolytic dosage of PGF,, on Day 7, GnRH 2 d later, and then time-inseminated approximately 16 h after the second treatment with G&II. Cows in Group 3 (n=83; abnormal, cystic) were treated with GnRH on Day 0, one luteolytic dosage of PGF,, on Day 7, and then inseminated at estrus observed within 7 d after the last treatment with PGF,,.

When cows were placed in the study (Day 0), parity (lactation number) and days in milk were recorded. Previous research (2,16) has shown that conception rates in this dairy herd were significantly higher during the period of October to December compared with other times of the year. Thus, the period of October to December was considered favorable, and the rest of the year was considered unfavorable for optimum pregnancy rates. The distribution of cows by parity, time of year (season), and days in milk for each group is shown in Table 1.

The methods of detection of estrus in this study included a computerized system which used increased walking activity and an accompanying decrease in milk production (AFIKIM system, Kibbutz Afikim 15 148, Israel) as well as daily visual observations of the cows. A specific individual was assigned the duty of observing the cows for estrus during each 8-h shift. Thus, these cows were continuously observed for estrus over an 8-h period of time for 24 h.

Pregnancy was determined by palpation of the uterus per rectum approximately 45 to 50 d after artificial insemination as previously described (28).

818 Theriogenology

Table 1. Distribution of cows by parity, season and days in milk for all groups Group 1 Group 2 n (%) n (%)

Parity 1 71 (35) 35 (47) 2 52 (26) 13 (17) 3+ 76 (38) 27 (36) Season October-December 63 (30) 24 (32) January-June 146 (70) 52 (68) Days in Milk 65-132 47 (23) 27 (36) 133-167 57 (28) 17 (22) 168-216 49 (24) 15 (20) 217-533 52 (25) 17 (22)

Group 3 n(%)

26 (31) 22 (27) 2.5 (42)

22 (27) 61 (73)

18 (22) 21 (25) 23 (28) 21 (25)

Data were obtained for the percentage of cows observed in estrus within 7 d after the last treatment with PGF,, (estrus detection rate), and conception rate to insemination at this time for cows in Group 3. Conception rate was defined as the percentage of cows that became pregnant divided by the total number of cows inseminated at estrus within 7 d after the last PGF,, treatment (Group 3 ), or when cows were time-inseminated (Groups 1 and 2).

Pregnancy rate to insemination at estrus within 7 d after PGF,, treatment (cows in Group 3) and to timed-insemination (cows in Groups 1 and 2) was determined. Pregnancy rate for cows in Group 3 was defined as the product of the estrus detection rate and the conception rate. For cows in Groups 1 and 2, conception rate and pregnancy rate were synonymous, and were considered to be conception as a result of timed-insemination (7).

The effects of different treatments on conception and pregnancy rates were compared using logistic regression (12) and were adjusted for parity, days in milk, and time of year (season). In this statistical model, the adjusted odds ratio (OR) indicates the probability of an event (conception/pregnancy) resulting from one treatment compared to that of another treatment and adjusting for different variables. Cows in Group 1 and Group 2 were compared to evaluate the effectiveness of synchronization of ovulation and timed-insemination in both normal and abnormal (cystic) cows. In addition, cows in Group 2 and Group 3 were compared to evaluate the effectiveness of synchronization of ovulation and timed-insemination and a regular protocol involving induction of estrus and insemination at detected estrus in cows with ovarian cysts.

Theriogenology

RESULTS

819

The descriptive statistics for estrus detection rate, conception rate and pregnancy rate for cows in all 3 groups are shown in Table 2.

Table 2. Estrus detection rate, conception rate and pregnancy rate for cows in all 3 groups n Estrus detection rate (%) Conception rate (%) Pregnancy rate (%)

Group 1 209 NA 31.5 31.5 2 76 NA 23.6 23.6 3 83 34.9 51.7 18 NA=not applicable

The effect of treatment (group) on conception/pregnancy rate for cows in Group 1 and Group 2, controlled for parity, time of year and days in milk, is shown in Table 3. The risk of nonconception/nonpregnancy was higher in Group 2 cows (OR=1.65) than in Group 1 cows but the difference was not significant (P=O. 11).

Table 3. Risk of nonconception or nonpregnancy in cows in Group 2 compared with cows in Group ( 1 ad’usted for arit ,

Adjusted odds ratio 95% CI P- value Group 1 1.0 2 1.65 0.89-3.08 0.11 Parity 1 1.0 2 1.32 0.65-2.69 0.45 3+ 1.06 0.58-1.95 0.84 Season October-December 1.0 January-May 1.64 0.89-3.03 0.11 Days in milk 65-129 1.0 130-165 1.29 0.59-2.84 0.53 166-215 0.90 0.41-1.98 0.80 216-533 0.77 0.35-1.68 0.51

820 Theriogenology

A comparison between normal cows subjected to timed-insemination and cystic cows subjected to timed-insemination and estrus synchronization and insemination at detected estrus is shown in Table 4. The risk of nonpregnancy was significantly higher (OR= 1.9) for cystic cows than for normal cows (P<O.Ol).

Table 4. Risk of nonpregnancy for cows in treatment Group 1, compared to cows in Groups 2 and 3 (adjusted for parity, season and days in milk)

Adjusted odds ratio 95% CI P- value Group 1 1.0 2813 1.9 1.10-3.10 <O.Ol Parity 1 1.0 2 1.1 0.60-2.00 0.66 3+ 0.7 0.40-I .30 0.33 Season October-December 1.0 January-May 1.5 0.90-2.70 0.09 Days in milk 65-129 1.0 130-165 1.4 0.70-2.80 0.33 166-215 1.0 0.50-2.10 0.94 216-533 0.8 0.40-l .70 0.69

The effect of treatment (group) on conception rate of cows in Group 2 and Group 3 adjusted for parity, time of year (season) and days in milk is shown in Table 5. The risk of nonconception was significantly higher in Group 2 (OR =3.58) than in Group 3 (P<O.Ol).

The effect of treatment (group) on pregnancy rate of cows in Group 2 and Group 3, controlled for parity, time of year and days in milk is shown in Table 6. The risk of nonpregnancy for cows in Group 3 (OR=l.37) was not significantly higher (P=O.45) than in Group 2.

Theriogenology 821

Table 5. Risk of nonconception in cows in Group 3 compared with cows in Group 2 (adjusted for parity, season and days in milk)

Adjusted odds ratio 95% CI P-value Group 3 1.0 2 3.58 1.38-9.27 0.01 Parity 1 1.0 2 0.77 0.22-2.72 0.69 3+ 0.58 0.14-1.39 0.29 Season October-December 1.0 January-May 1.42 0.54-3.70 0.47 Days in milk 65-129 1.0 130-165 0.72 0.22-2.40 0.59 166-215 1.68 0.44-6.40 0.45 216-533 0.73 0.22-2.38 0.60

Table 6. Risk of nonpregnancy in cows in treatment Group 3 compared with cows in Group 2 (adjusted for parity, season and days in milk)

Adjusted odds ratio 95% CI P-value Group 2 1.0 3 1.37 0.61-3.08 0.45 Parity 1 1.0 2 0.76 0.25-2.27 0.62 3+ 0.51 0.21-1.27 0.15 Season October-December 1.0 January-May 1.59 0.69-3.70 0.28 Days in milk 65-129 1.0 130-165 1.49 0.50-4.44 0.47 166-215 2.15 0.63-7.31 0.22 216-533 1.04 0.36-3.01 0.95

822

DISCUSSION

Theriogenology

While there are numerous-reports on the effectiveness of using synchronization of ovulation and timed-insemination on the reproductive performance of normal, cyclic dairy cows, there are no scientific studies on the effectiveness of using this treatment strategy for bovine ovarian cysts. Nevertheless, there have been numerous reports from veterinary practitioners of the success of this treatment strategy for cystic cows (27).

This study was designed to compare the effectiveness of the synchronization of ovulation with timed-insemination and induction of estrus with insemination at estrus in the treatment of bovine ovarian cysts. The rationale was that the hormones used to synchronize ovulation in normal dairy cows are the same as those used to treat bovine ovarian cysts. In addition, cows can be artificially inseminated following treatment without the need for detection of estrus (timed- insemination).

It has been reported that nearly half of the estrous periods in normal, cyclic dairy cows may not be detected (3,19,22). In our present study, the rate of estrus detection in Group 3 cows was only 35% (29/83). This value was significantly lower than that reported in a previous study using cows in this same dairy herd (16). While accuracy of estrus detection may have a significant effect on the rate of detection, the present study was not designed to evaluate this variable. Nevertheless, the estrus detection rate in our study was extremely low, particularly considering the fact that 2 different estrus detection methods (visual observation and a computerized method) were used in the herd. It is speculated that the low rate of detection in our present study could be associated with the use of bovine somatotropin, which was more widespread and involved all the cows in the milking herd unlike that in a previous study (16).

Previous research (14,15,17) has shown an inhibitory effect of bST on the expression and intensity of estrus in dairy cows. This effect was also shown in steroid-primed, ovariectomized heifers (15). In one of these studies (14), follicular turnover was more rapid and concentrations of estradiol increased earlier after PGF, administration in cows treated with bST, but the absolute concentrations of progesterone and estradiol were not different between treatments. It was therefore assumed that differences in steroid concentrations were not a causative factor in the lack of expression of estrus in cows treated with bST (14). It has also been suggested that there may be direct effects of bST on behavioral centers in the brain (14). While the present study was not designed to evaluate the effect of bovine somatotropin on the expression of estrus in the dairy cow, the collective evidence in the literature could support the speculation that the low rate of estrus detection observed in this herd was probably causally associated with the use of bovine somatotropin.

Bovine ovarian cysts have traditionally been classified as follicular or luteal, based on clinical examination by per rectum palpation of the ovaries. In this study, no attempt was made

Theriogenology 823

to distinguish between follicular and luteal cysts in the cows, because previous reports (10,24) have shown the inadequacy of palpation of the ovaries per rectum to make this distinction. In addition, because of the large number of cows used, it was not practical to use ultrasonography.

Nevertheless, it did not appear necessary to make this distinction between follicular and luteal cysts, especially in light of the current concept of the etiology, turnover rate and fate of bovine ovarian cysts (11). The essence of this concept is that cows with bovine ovarian cysts (anovulatory follicular structures) have ovarian follicular waves similar to those of cows which ovulate (normal cows) at the end of es&us, but these follicles fail to ovulate. In addition, these anovulatory ovarian structures eventually regress, and may be replaced by similar anovulatory follicular structures if the cow continues to experience an absence of release of G&I-I to initiate ovulation. This concept also states that these anovulatory follicular structures do not ovulate in response to exogenous GnRH, but treatment with GnRH causes ovulation of a mature follicle of an ovarian follicular wave. The resulting CL then undergoes luteolysis, and normal cyclicity is restored.

In this study, the criterion used to determine a successful response of cystic cows to treatment with GnRH followed by one luteolytic dosage of PGF,, (cows in Group 3) was the occurrence of estrns withii 7 d after the last PGF,, treatment. Therefore, only cows which were observed in es&us and inseminated were used to determine conception rate for cows in Group 3.

Conception/pregnancy rate using a protocol involving synchronization of ovulation and timed-insemination (Ovsynch) was 3 1.5% for normal cows (Group 1) and 23.6% for abnormal cystic cows (Group 2). These values were not significantly different (P~0.10). However, the pregnancy rate for normal cows (Group 1) was higher (PcO.01) than for cystic cows (Groups 2 and 3). In this comparison it should be considered that cystic cows were subjected to 2 different treatment schemes (timed-insemination and insemination at an induced estrus). In the present study, the conception/pregnancy rate using timed-insemination in normal cows (3 1.5%) was similar to those of previously published studies in Florida (7, 16).

When cows in Group 3 were observed in estrus and inseminated within 7 d after the last treatment with PGF,,, the conception rate (5 1.7%) was higher than that observed for cows in Group 2 (23.6%). However, due to the poor estrus detection rate (34.9%), the pregnancy rate of cows in Group 3 (18%) was not higher than that observed for cows in Group 2 (23.6 %).

Therefore, the effectiveness of treating cystic cows using a protocol involving GnRH, PGF,,, and timed-insemination compared with one using GnRH and PGF,, varies depending upon the criterion used to determine success of treatment (conception rate or pregnancy rate). However, economic reality dictates that the pregnancy rate be a more important criterion for evaluation of success of treatment than the conception rate, because it includes all the cows in the group, and the estrus detection rate was low in this herd.

824 Theriogenology

Previous research in this dairy herd (16) demonstrated that the time of the year had a significant effect on the conception rate and pregnancy rate following the use of PGF2,, and insemination at induced estrus or timed-insemination using PGF,,, and GnRH. However, in the present study, no effect of time of the year was observed. This is probably related to the fact that the present study was conducted during the period of September to June, and did not include the months of July and August as did the previous study.

In conclusion, a similar pregnancy rate was observed in cystic cows subjected to synchronization of ovulation and timed-insemination (Group 2), and cystic cows subjected to synchronization of estms and insemination at estrus within 7 d after injection of PGF,, (Group 3). The benefit of using timed-insemination in lactating dairy cows with ovarian cysts lies in the fact that these cows do not have to be detected in estrus for insemination and achieving pregnancy.

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