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Reproductive performance of dairy cows with ovarian cysts after different GnRH and cloprostenol treatments
Theriogenology 58 (2002) 1337±1348
Reproductive performance of dairy cows with ovarian cysts after different GnRH and cloprostenol treatments F. LoÂpez-Gatiusa,*, M. LoÂpez-BeÂjarb a
Departamento de Produccion Animal, Escuela TeÂcnica Superior de IngenierõÂa Agraria, Universidad de Lleida, Avda. Alcalde Rovira Roure 177, 25198 Lleida, Spain b Department of Veterinarian Anatomy and Embryology, Autonomous University of Barcelona, Barcelona, Spain Received 1 October 2001; accepted 14 February 2002
Abstract Cystic ovarian disease is an important cause of reproductive failure and economic loss for the dairy industry. This report describes two consecutive studies. The objective of the ®rst was to evaluate the response of cows with ovarian cysts to two therapeutic treatments. In the second study, we compared the effectiveness of the best treatment established in Study 1 with that of the Ovsynch protocol. For Study 1, cows were considered to have an ovarian cyst if it was possible to observe a single follicular structure with a follicular antrum diameter 25 mm in the absence of a corpus luteum in three ultrasonographic examinations performed at 7 days intervals. At diagnosis (Day 0), cows were assigned to one of two treatment groups. Cows in Group GnRH/CLP (n 31) were treated with 100 mg GnRH i.m. and 500 mg cloprostenol (CLP) i.m. on Day 14. Cows in Group GnRH±CLP/CLP (n 32) were treated with 100 mg GnRH i.m. plus 500 mg CLP i.m. on Day 0, and 500 mg CLP i.m. on Day 14. The animals were inseminated at observed estrus and monitored weekly by ultrasonography for 4 weeks or until AI. Cows in the GnRH±CLP/CLP group showed a lower cystic persistence rate (15:6% < 45:2%; P 0:01); a higher estrus detection rate (84:4% > 41:9%; P < 0:0001); a higher ovulation rate (75% versus 32.3%; P < 0:0001) and a higher early response rate (31% > 3%; P 0:02) than those in the GnRH/CLP group. For the second study, 128 cows with ovarian cysts were randomly assigned to one of two treatment groups: cows in Group Ovsynch (n 64) were treated with 100 mg GnRH i.m. on Day 0, 500 mg CLP on Day 7, and 100 mm GnRH i.m. 36 h later. Cows in this group were inseminated 24 h after the second GnRH dose (Ovsynch protocol). Cows in Group GnRH±CLP/CLP/GnRH (n 64) were treated as those in the GnRH±CLP/CLP group of Study 1 but received GnRH 32 h after the second CLP treatment and were inseminated 24 h after this. A further group of cows without ovarian cysts inseminated at natural estrus served as the Group Control (n 64). Cows in the GnRH±CLP/CLP/ GnRH group showed a lower cystic persistence rate (10:9% < 46:9%; P < 0:0001); higher ovulation * Corresponding author. Tel.: 34-973-702-500; fax: 34-973-238-264. E-mail address: [email protected] (F. LoÂpez-Gatius).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 9 5 2 - 4
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rate (79:7% > 17:2%; P < 0:0001); higher return to estrus rate (34:3% > 12:5%; P < 0:01) and higher pregnancy rate (28:1% > 3:1%; P < 0:01) than those in Ovsynch; and a similar pregnancy rate (28.1% versus 35.9%) to Control cows. These ®ndings indicate that lactating cows with ovarian cysts can be successfully synchronized and time inseminated using a protocol that combines GnRH and CLP, starting treatment by simultaneously administering both products. This protocol also allows the insemination of cows showing estrus within the ®rst week of treatment. Ovarian cysts were less responsive when treatment was started with GnRH alone. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Dairy cows; Ovarian cysts; GnRH; Cloprostenol
1. Introduction Ovarian cysts are anovulatory follicle-like structures >2.5 cm that persist for 10 days or more in the absence of a corpus luteum [1]. Cystic ovarian disease is a common ovarian disorder diagnosed in dairy cattle and is an important cause of reproductive failure and economic loss for the dairy industry. Depending on plasma progesterone concentration, ovarian cysts can be classi®ed as follicular (low progesterone, 30±85%) or luteal (high progesterone, 15±70%) [2]. Follicular cysts usually have a thin wall and are ¯uctuant on palpation, whereas luteal cysts have a thick wall with luteal tissue and are hard when palpated [3]. The highest incidence rates of cysts have been reported for the intervals before 60 days in milk [4] and between 14 and 40 days postpartum [5]. Follicular cysts, more frequent than luteal cysts during the early postpartum period, progress to become luteal cysts through the luteinization process and ®nally regress [6]. In fact, cysts that develop during the early postpartum period regress spontaneously in more than 50% of cows before insemination programs commence [7]. However, since there is a chance of persistence of cysts beyond 60 days postpartum it may be better to start the treatment immediately rather than waiting for spontaneous recovery [8]. Since the 1970s, hCG and GnRH analogues have been used to treat ovarian cysts. Both hCG and GnRH appear to be equally effective with respect to treatment response and posttreatment fertility [7]. Concerning administration of GnRH, within 28±30 days after a GnRH treatment, 65±80% of cows with ovarian cysts re-establish ovarian cycles [9]. Cysts respond to GnRH by luteinization of the cystic structure [10]. However, luteal function is not always re-established after treatment of follicular cysts with GnRH [11]; Sprecher et al. reported that luteal cysts failed to respond to GnRH during 10 days of treatment [12]. Another option for treatment of cystic ovaries is prostaglandin F2a and its synthetic analogues. Prostaglandins act faster than GnRH: within 2±5 days in over 90% of cases they cause lysis of luteal cysts, either luteinized spontaneously or following GnRH treatment, resulting in normal estrus and fertility [11]. Nevertheless, the response to prostaglandins shown by cows with follicular cysts is poor [13]. Thus, the nature of the cyst needs to be determined before appropriate treatment may be prescribed. The response of cows with ovarian cysts to speci®c GnRH or prostaglandin therapy assigned by a milk progesterone assay has been demonstrated to be much better than treatment with GnRH alone (72% versus 52%) [12]. However, milk progesterone tests are not extensively available, and it is often dif®cult to differentiate luteal from follicular cysts under farm conditions. It is for this
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reason that GnRH is widely used to treat both types of cyst, and prostaglandins used to shorten the interval from GnRH treatment to estrus. Thus, treatment of ovarian cysts with GnRH and a luteolytic agent 9±14 days later, is considered an ef®cient method of cyst recovery without the need to distinguish the two types of ovarian cyst [1,2,7,9]. If all cysts are simultaneously treated with both GnRH and prostaglandins, it would not matter whether the cyst was luteal or follicular, and the interval from treatment to estrus, at least in luteal cysts, would be reduced. In an attempt to treat cystic ovaries using a combination of GnRH and cloprostenol (CLP), combined treatment appeared to have no advantage over the use of GnRH alone, however, prostaglandin was not administered after treatment to shorten the time to estrus [14]. The success of cyst therapy depends mostly on an ef®cient detection of estrus. If estrus is not detected, treated cows cannot be inseminated. Thus, a therapeutic protocol for timedinsemination in cows with ovarian cysts would be of great interest. In a recent study [15], it was suggested that synchronization of ovulation and timed-insemination using the Ovsynch protocol [16] may be an effective method of establishing pregnancy in lactating dairy cows with ovarian cysts. On these grounds, we undertook two consecutive studies on cows which had completed the voluntary waiting period (about 60 days postpartum). The objective of the ®rst study was to evaluate the response shown by cows with ovarian cysts to two treatments: GnRH on Day 0 and CLP administered 14 days later; or simultaneous treatment with GnRH and CLP on Day 0, followed by CLP on Day 14. The second study was designed to compare the reproductive performance after timed AI of cows with ovarian cysts that were treated with GnRH and CLP on Day 0, CLP on Day 14 and GnRH 36 h later, with that shown by animals treated with GnRH on Day 0, CLP on Day 7 and GnRH 36 h later (Ovsynch protocol). 2. Materials and methods 2.1. Study 1 2.1.1. Animals This study was performed on a commercial dairy herd of 650 mature cows in northeastern Spain over the period July 1998 to June 1999. The voluntary waiting period from calving to ®rst AI established for this dairy herd was 60 days. The study population was formed by 63 cows in their ®rst or second lactation period. Mean daily milk production of these cows 10 days before inclusion in the study was 48 kg. The cows were milked three times daily and kept in open stalls. Cows with clinical disorders detected on postpartum examination or during the study were withdrawn from the program. Excluding disorders were: mastitis, lameness, digestive disorders, abnormal genital discharges and pathological abnormalities of the reproductive tract detectable on palpation per rectum. The herd was maintained on a weekly reproductive health program. The reproductive tract of each animal was examined by palpation per rectum within 29±35 days postpartum to check for normal uterine involution and ovarian structures. For ultrasonographic examination, we selected cows with a single follicular structure estimated to be greater than 25 mm, no corpus luteum, and no estrus signs in the previous 14 days. These animals
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were then subjected to three ultrasonographic examinations at intervals of 7 days, starting 14 days after the postpartum examination. A cow was considered to have a ovarian cyst when a follicular structure (of antrum diameter larger than 25 mm) could be observed in all the ultrasonographic examinations in the absence of a corpus luteum (Fig. 1a). 2.1.2. Ultrasonography Cows were diagnosed as having an ovarian cyst by transrectal ultrasonography using a portable B-mode ultrasound scanner (Scanner 100 Vet, equipped with a 5.0 MHz transducer; Pie Medical, Maastricht, The Netherlands). Each ovary was scanned in several planes by moving the transducer along its surface to identify the different structures. The size of cysts was measured using the built in electronic caliper after freezing the image on screen. The largest and the smallest diameters of the follicular antrum were measured and the mean diameter was then recorded. Cows found to have ovarian cysts were subjected to ultrasonographic examinations weekly for 4 weeks or until AI. Ultrasonography was also performed on the ovaries of inseminated cows 11 days after AI. All examinations were performed by the same operator. Cyst regression was registered when a clear decrease of the follicular antrum diameter to a value lower than 20 mm was observed by ultrasound (Fig. 1b). 2.1.3. Treatments and arti®cial insemination At the time of diagnosis of ovarian cysts (Day 0), the cows were alternately assigned to one of two treatment groups (Fig. 2) according to the chronological order of their calving data. Cows in Group GnRH/CLP (n 31) were treated with GnRH (100 mg, i.m.; Dalmarelin, FATRO, Bologna, Italy) on Day 0, and given one luteolytic dose of CLP (500 mg; Estrumate, Schering Plough Animal Health, Madrid, Spain) on Day 14. Cows in Group GnRH±CLP/CLP (n 32) received GnRH plus CLP on Day 0, and CLP on Day 14. All animals were inseminated at observed estrus from Day 0 by the same practitioner using frozen semen from a single ejaculate. 2.1.4. Progesterone analysis Blood samples were taken at the beginning of treatment (Day 0), and 11 days after AI in inseminated cows. All blood was collected from the coccygeal vein into heparinized vacuum tubes. Plasma was separated by centrifugation within 2 h and stored at 20 8C until assayed. Progesterone was determined using solid-phase RIA kits containing antibody-coated tubes, 125 I-labeled progesterone and rabbit antiserum (CS Bio International, Gif-Yvette, France). The RIA method was previously validated for its use in the cow as described by Guilbault et al. [17]. The sensitivity of the assay was 0.05 ng/ml progesterone. Plasma samples showing hormone concentrations below this were assigned the sensitivity value. The intra-assay coef®cient of variation was 16%. Plasma progesterone concentrations were used to classify cysts as luteal (1 ng/ml) or follicular (<1 ng/ml). 2.1.5. Detection of estrus, ovulation and pregnancy diagnosis The animals were inspected for signs of estrus (standing to be mounted) at least four times throughout the day. Cows were inseminated approximately 8±10 h after the ®rst signs of estrus were observed. Ovulation was de®ned as the presence of a corpus luteum and
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Fig. 1. Selected ultrasound pictures of a follicular cyst and its regression from a cow which became pregnant, in the GnRH±CLP/CLP/GnRH group (Study 2): (a) follicular cyst (treatment Day 0); (b) regressed cyst (treatment Day 14); (c) corpus luteum 11 days after AI. All images were from the right ovary. Bar: 20 mm.
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Fig. 2. Schematic summary of treatment groups, ultrasonographic examinations and blood sampling times.
plasma progesterone levels 1 ng/ml 11 days after insemination. A corpus luteum was identi®ed by its size and shape as well as by a granular, gray, oval area, that could be delineated from the remaining ovarian tissue (Fig. 1c) [18]. Pregnancy diagnosis was performed by ultrasonography at 34±40 days postinsemination. 2.1.6. Data analysis The cystic persistence rate was de®ned as the percentage of cows maintaining a follicular structure with an antrum diameter 25 mm in the absence of a corpus luteum on Day 28 after starting treatment. The estrus detection rate was taken as the percentage of cows showing estrus, expressed as a percentage of the total number of cows in each treatment group, between Days 0 (starting treatment) and 28. The ovulation rate was the percentage of inseminated cows that ovulated, expressed as a percentage of the total number of cows in each group at the same period. The early response rate was de®ned as the number of animals that showed estrus and ovulated before ®nishing treatment, as a percentage of the total number of cows in each group between Days 0 (starting treatment) and 14. The pregnancy rate was taken as the number of cows inseminated between Days 0 and 28, that became pregnant, expressed as a percentage of the total number of cows in each group. Treatment regimes were compared in terms of cyst persistence, estrus detection, ovulation, early response and pregnancy rates using the Chi-square test. 2.2. Study 2 2.2.1. Animals, treatments and timed insemination The second study was performed in the same dairy herd over a 14-month-period (1 January 2000 to 28 February 2001). The cows were subjected to the same reproduc-
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tive program and ovarian cysts were diagnosed by ultrasonography as described for Study 1. The study population was formed by 192 cows: 128 cows with ovarian cysts free of other clinical disorders, and 64 healthy animals used as controls. The lactation number was 2.8 and ranged from 1 to 11 lactations. Mean daily milk production 10 days before recruiting for the study was 54 kg. Cows with ovarian cysts were paired by lactation number as ®rst, second, or third or more lactations. One cow from each pair was alternately assigned, according to the chronological order of their calving data, to receive one of two treatments: the Ovsynch program designed by Pursley et al. [16]; or GnRH plus CLP on Day 0 followed by CLP on Day 14, as described in Study 1, plus GnRH 36 h later to synchronize ovulation (Group GnRH±CLP/CLP/GnRH; n 64). Cows in Group Ovsynch (n 64) were treated with GnRH (100 mg i.m.; Dalmarelin) on Day 0 and were given one luteolytic dose of CLP (500 mg i.m.; Estrumate) on Day 7, and 100 mm GnRH i.m. 32 h later. The cows were then inseminated 24 h after the second GnRH treatment in both groups. Thus, including diagnosis and treatment times, cows with ovarian cysts were inseminated between 74 and 80 days after parturition. Ovulation was con®rmed by ultrasonography 11 days after insemination by the presence of a corpus luteum. To assess their subsequent fertility, cows with ovarian cysts were paired with healthy animals (Group Control; n 64) of the same lactational period, inseminated at natural estrus during the same postpartum interval. All animals were inseminated by the same practitioner with frozen semen from a single bull of proven fertility. Cows with ovarian cysts were inseminated without visual detection of estrus, and control cows were inseminated approximately 8±10 h after the ®rst signs of estrus were observed. 2.2.2. Collection of data after insemination Cows returning to estrus from Days 8 to 30 postinsemination were reinseminated. Cows that exhibited estrus after this interval and before pregnancy diagnosis were not inseminated. Pregnancy was diagnosed by ultrasonography at 42 days (Ovsynch group) and at 35 days (GnRH±CLP/CLP/GnRH group) postinsemination. Thus, pregnancy diagnosis was performed 52 days after starting treatment for both groups. Non-pregnant cows with a follicular structure of antrum diameter larger than 25 mm that lacked a corpus luteum, were subjected to a further ultrasonographic examination the following week to determine the presence of an ovarian cyst. 2.2.3. Data analysis The effect of treatment was evaluated in terms of ovulation rate (number of cows with at least one corpus luteum on Day 11 after AI as a percentage of the total number of cows in each group), pregnancy rate (number of pregnant cows after ®rst AI as a percentage of the total number of cows in each group), cows returning to estrus (number of cows returning to estrus 8±30 days postinsemination), and cystic persistence rate (number of cows with an ovarian cyst 7 days after pregnancy diagnosis as a percentage of the total number of cows in each group). Treatment regimes were compared using the Chi-square test. Values corresponding to both studies are expressed as the mean S:D:
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3. Results Throughout both studies, no short cycles or episodes of nymphomania were registered. Selected pictures of a follicular cyst and its evolution from a cow which became pregnant, in the GnRH±CLP/CLP/GnRH group (Study 2), are shown in Fig. 1. 3.1. Study 1 The mean number of days from calving to diagnosis of ovarian cysts (Day 0) was 59:2 1:9 days and ranged from 57 to 63 days. Twenty-six cows (41.3%) had luteal and 37 (58.7%) follicular cysts (plasma progesterone concentration 1 and <1, respectively). Table 1 shows the distribution of the type of cyst per treatment. In the GnRH/CLP group, of 19 cows with previous follicular cysts, 1 cow showed estrus signs within the ®rst week after GnRH treatment, and 9 cows showed estrus within the ®rst week after CLP treatment. Three cows became pregnant. Cystic structures regressed in 1 cow by Day 7 and in 10 cows by Day 14. One cow showing cystic regression on Day 14 developed a new cyst before Day 28. Of the 12 cows with previous luteal cysts, no regression of cystic structures was registered on Day 7 and cysts regressed in 5 cows by Day 14. These 5 cows showed no signs of estrus after CLP treatment. Three of the remaining 9 cows with luteal cysts showed estrus following CLP treatment. One of these 3 cows became pregnant. In the GnRH±CLP/CLP group, three cows with previous follicular cysts and seven with previous luteal cysts showed estrus signs within the ®rst week of GnRH±CLP treatment. Three of these cows became pregnant. Ten cows with previous follicular cysts and seven cows with previous luteal cysts showed estrus during the ®rst week after the second CLP treatment. Six of these cows became pregnant. Of the 18 cows with previous follicular cysts, cystic structures regressed in 3 cows by Day 7 and in 11 cows by Day 14. Of the Table 1 Effect of treatment with GnRH/CLP or GnRH±CLP/CLP 28 days after a diagnosis of ovarian cysts1 Treatment group
n
Type of cyst
Cystic persistence (%)
Estrus detection (%)
Ovulation (%)
Early response (%)
Pregnancy (%)
GnRH/CLP2
19 12 31
Follicular Luteal Total
11/19 (58)a 3/12 (25) 14/31 (45)b
10/19 (53) 3/12 (25)e 13/31 (42)f
7/19 (37) 3/12 (25)I 10/31 (32)j
1/19 (5) 0/12 (0) 1/31 (3)m
3/19 (16) 1/12 (8) 4/31 (13)
GnRH±CLP/CLP3
18 14 32
Follicular Luteal Total
4/18 (22)c 1/14 (7) 5/32 (16)d
13/18 (72) 11/18 (61) 14/14 (100)g 13/14 (93)k 27/32 (84)h 24/32 (75)l
3/18 (17) 4/18 (22) 7/14 (50) 5/14 (36) 10/32 (31)n 9/32 (28)
Different superscripts within the same column denote signi®cant differences detected by the Chi-square test (a, c: P 0:03; m, n: P 0:02; b, d: P 0:01; e±l: P < 0:0001). 1 Results were determined in terms of cystic persistence, estrus, ovulation, early response and pregnancy rates. Percentages were obtained from total number of animals (n) in each group. 2 GnRH/CLP 100 mg GnRH i.m. on Day 0, and 500 mg CLP i.m. on Day 14. 3 GnRH±CLP/CLP 100 mg GnRH i.m. plus 500 mg CLP i.m. on Day 0, and 500 mg CLP i.m. on Day 14.
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Table 2 Effect of treatment regimen on ovulation rate, percentage of cows returning to estrus, pregnancy rate and percentage of non-pregnant cows with ovarian cysts 7 days after pregnancy diagnosis Effects
Control (n 64)
Ovsynch1 (n 64)
GnRH±CLP/CLP/GnRH2 (n 64)
Ovulation rate (%) Returns to estrus (%) Pregnancy rate (%) Cystic persistence (%)
± 21/64 (33)c 23/64 (36)f ±
11/64 (17)a 8/64 (13)d 2/64 (3)g 30/64 (47)i
51/64 (80)b 22/64 (34)ce 18/64 (28)fh 7/64 (11)j
Different superscripts within the same row denote signi®cant differences detected by the Chi-square test (c, d, ce, g, fh: P < 0:01; a, b, f, g, i, j: P < 0:0001). 1 Group Ovsynch 100 mg GnRH i.m. on Day 0; 500 mg CLP i.m. on Day 7 followed by 100 mm GnRH i.m. 32 h later; AI 24 h after the second GnRH treatment. 2 Group GnRH±CLP/CLP/GnRH 100 mg GnRH i.m. plus 500 mg CLP i.m. on Day 0, 500 mg CLP i.m. on Day 14 followed by 100 mm GnRH i.m. 32 h later; AI 24 h after the second GnRH treatment.
14 cows with luteal cysts, cystic structures regressed in 10 cows by Day 7 and in 4 cows by Day 14. Ovulation was con®rmed 11 days after AI by the presence of a corpus luteum and luteal activity in 10 of 13 inseminated cows in the GnRH/CLP group, and in 24 of 27 inseminated cows in the GnRH±CLP/CLP group. Four of the cows that failed to ovulate developed cysts once again and were diagnosed as having persistent cysts: two cows with previous follicular cysts in the GnRH/CLP group, and one cow with previous follicular and another with previous luteal cysts in the GnRH±CLP/CLP group. All 11 cows showing estrus within the ®rst week after the onset of treatment ovulated. Table 1 shows the persistent cyst, estrus detection, ovulation, early response and pregnancy rates for cows in the two treatment groups. A signi®cant effect by treatment was shown on persistent cyst, estrus detection, ovulation and early response rates. Pregnancy rates were not signi®cantly affected. 3.2. Study 2 The mean number of days from calving to diagnosis of ovarian cysts (Day 0) was 60:5 2:1 days, ranging from 57 to 63 days. A signi®cant effect by treatment was shown on the ovulation, return to estrus and pregnancy rates, and on the percentage of open cows with ovarian cysts 7 days after pregnancy diagnosis (Table 2). 4. Discussion Study 1 shows the effectiveness of GnRH plus CLP and CLP administered 14 days later in resolving cysts without the need for diagnosing the type of ovarian cyst. The cystic persistence rate (16%) was signi®cantly lower than that observed using GnRH alone plus CLP 14 days later (45%). Furthermore, estrus detection and ovulation rates were signi®cantly higher in response to GnRH±CLP/CLP treatment. In this last treatment
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group, 10 cows (31%) showed estrus and ovulated within the ®rst week after treatment onset, compared to 1 cow (3%) in the GnRH/CLP group. This ®nding is of considerable interest for dairy farms with an ef®cient estrous detection system: it is suggested that using the GnRH±CLP/CLP protocol, a proportion of cows could be inseminated before ®nishing treatment, thus reducing the number of open days. Follicular cysts showed a worse recovery response in the GnRH/CLP than in the GnRH± CLP/CLP group (42% versus 78% regression, respectively). These results indicate a possible synergistic effect of CLP on GnRH in luteinizing follicular cysts. Exogenous prostaglandins can induce a hypophysial LH release [19]. Conversely, GnRH does not appear to inhibit the luteolytic effect of CLP on cows with luteal cysts, as previously reported [14]. Seven of 14 cows with luteal cysts in the GnRH±CLP/CLP group showed estrus and ovulated within the ®rst week after GnRH±CLP treatment. However, in the GnRH/CLP group, no cows with luteal cysts showed estrus before Day 14. A poor response or lack of response of luteal cysts to GnRH has also been previously reported [11,12]. No cystic regression was registered by ultrasonography on Day 7 of treatment in cows with luteal cysts, and 5 of 12 cows, in which cysts regressed 14 days after GnRH treatment, were not responsive to subsequent CLP. It is likely that the effect of prostaglandins on luteal cysts following GnRH treatment could be improved by reducing the interval from GnRH to prostaglandin treatment. Our Study 1 results differ in part from those of Dinsmore et al. [14], in which simultaneous administration of GnRH and CLP appeared to have no advantage over GnRH alone. In our study, the administration of CLP 14 days after treatment probably had the effect of inducing estrus in more cows, leading to differences between treatments. The present study was performed on a single dairy herd, eliminating the herd effect, to ensure that animals receiving different treatment regimes were subjected to the same management practices. Dinsmore et al. [14] performed their study on 75 cows from 32 herds, a factor that may have masked ovarian cystic responses to treatments. Based on the ®ndings of Study 1, the GnRH±CLP/CLP treatment was used in the second study which involved the administration of GnRH 36 h after the second CLP treatment, to synchronize ovulation. We compared the reproductive performance of lactating dairy cows subjected to the Ovsynch protocol (Ovsynch group) with that of cows simultaneously administered GnRH and CLP on Day 0, CLP 14 days later and GnRH 36 h after this (GnRH±CLP/CLP/GnRH group), followed by timed insemination. Best results were associated with GnRH±CLP/CLP/GnRH treatment, with a higher ovulation rate (80%), return to estrus rate (34%) and pregnancy rate (28%) recorded, along with a lower cystic persistence rate (11%) 52 days after starting treatment. For cows in this group, similar return to estrus and pregnancy rates were recorded after timed insemination for noncystic (33 and 36%, respectively) and cystic cows (34 and 28%, respectively). While this work was in progress, a paper by Bartolome et al. was published [20], in which the authors reported that using the Ovsynch protocol as a therapeutic strategy for bovine ovarian cysts in dairy cows, similar pregnancy rates were obtained after timed insemination in cows with (23.6%) and without (31.5%) cysts. Our results are in clear disagreement: only 2 out of 64 cows with cysts (3%) became pregnant following the Ovsynch protocol plus timed insemination. The explanation for this discrepancy could be related to the variable ``number of days in milk''. In the study by Bartolome et al. [20], days in milk ranged from 65 to 533 days
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(over 75% of cows ranged from 133 to 533 days), a period of positive energy balance for most cows [21]. Our study was conducted during a short postpartum period; ovarian cysts were diagnosed within 57±63 days after calving (a common voluntary waiting period from calving to ®rst insemination). During this period, the metabolic demands of lactation often hinder ®nal follicular maturation and ovulation. It is also possible that cows with ovarian cysts were recovering from extended periods of negative energy balance after calving. The predominance of follicular cysts registered in Study 1 (59% follicular versus 41% luteal) could be accounted for by the metabolic status of the cows. Ovarian cysts are dynamic in nature, and cysts classi®ed as follicular may be later classi®ed as luteal during their lifespan [1,7]. From a clinical perspective, we might consider follicular cysts as a ®rst stage in the ovarian cystic condition, and luteal cysts as a sign of recovery. With these points in mind, we may propose that when cows enter the positive balance period 90±120 postpartum, as the number of days in milk increases, the probability of a predominance of luteal cysts also rises. If so, the Ovsynch protocol would be more ef®cient in advanced lactation periods in which luteal cysts predominate. As noted above in Study 1, prostaglandins should be more ef®cient if applied before Day 14 after GnRH treatment in cows with luteal cysts. On the contrary, when prostaglandins are administered on Day 7 (Ovsynch protocol), follicular cysts have too short a period for recovery. Nanda et al. [22] demonstrated that luteinized follicular cysts showed a poor response to prostaglandins administered 7 days after GnRH treatment. Luteal tissue producing progesterone for less than 7 days after GnRH treatment in cows with follicular cysts may be slightly responsive to prostaglandins, perhaps as occurs in the early luteal period of cyclic cows [23]. Corpus luteum refractoriness to prostaglandin F2a during the early bovine luteal phase has been partly related to the fact that neovascularization is as yet incomplete [24]. We recently demonstrated that neovascularization in the luteinized area of cysts in rabbits occurs only at advanced stages of cystic luteinization, with endothelial cells remaining near the basament membrane area of the cysts during the early cystic luteinization process [25]. In conclusion, the present ®ndings indicate that lactating cows with ovarian cysts can be successfully synchronized and time inseminated using a protocol that combines GnRH and CLP, starting treatment by the simultaneous administration of both products. This protocol also allows cows showing estrus within the ®rst week of treatment to be inseminated in farms with an ef®cient estrus detection system. In contrast, ovarian cysts are less responsive to starting treatment with GnRH alone. Acknowledgements The authors thank Ana Burton for assistance with the English translation. This study received ®nancial support from the CTT of University of Lleida (C-0243 with Schering Plough Animal Health, Spain). References [1] Garverick HA. Ovarian follicular cysts in dairy cows. J Dairy Sci 1997;80:995±1004. [2] Farin PW, Estill CT. Infertility due to abnormalities of the ovaries in cattle. Food Anim Pract 1993;9:291±308.
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[3] Seguin B. Ovarian cysts in dairy cows. In: Morrow DA, editor. Current therapy in theriogenology. Philadelphia: WB Saunders, 1980. p. 199±204. [4] Erb HN, White ME. Incidence rates of cystic follicles in Holstein cows according to 15-day and 30-day intervals. Cornell Vet 1981;71:326±31. [5] Morrow DA, Roberts SJ, McEntee K. A review of postpartum ovarian activity and involution of the uterus and cervix in cattle. Cornell Vet 1969;59:134±54. [6] Carroll DJ, Pierson RA, Hauser ER, Grummer RR, Combs DK. Variability of ovarian structures and plasma progesterone pro®les in dairy cows with ovarian cysts. Theriogenology 1990;34:349±70. [7] Peter AT. Infertility due to abnormalities of the ovaries. In: Youngquist RS, editor. Current therapy in large animal theriogenology. Philadelphia: WB Saunders, 1997. p. 349±54. [8] White ME, Erb H. Treatment of ovarian cysts in dairy cattle. A decision analysis. Cornell Vet 1980;70:247±57. [9] Ijaz A, Fahning ML, Zemjanis R. Treatment and control of cystic ovarian disease in dairy cattle: a review. Br Vet J 1987;143:226±37. [10] Kesler DJ, Elmore RG, Brown EM, Garverick HA. Gon, Gonadotropin releasing hormone treatment of dairy cows with ovarian cysts. I. Gross ovarian morphology and endocrinology. Theriogenology 1981;16:207±17. [11] Dobson H, Ranking JEF, Ward WR. Bovine cystic ovarian disease: plasma hormone concentrations and treatment. Vet Rec 1977;101:459±61. [12] Sprecher DJ, Strelow LW, Nebel RL. The response of cows with cystic ovarian degeneration to luteotropic or luteolytic therapy as assigned by latex agglutination milk progesterone assay. Theriogenology 1990;34:1149±58. [13] Leslie K, Bosu WTK. Plasma progesterone concentrations in dairy cows with cystic ovaries and clinical responses following treatment with fenprostalene. Can Vet J 1983;24:352±6. [14] Dinsmore RP, White M, English PB. An evaluation of simultaneous GnRH and cloprostenol treatment of dairy cattle with cystic ovaries. Can Vet J 1990;31:280±4. [15] Fricke PM, Wiltbank MC. Effect of milk production on the incidence of double ovulation in dairy cows. Theriogenology 1999;52:1133±43. [16] Pursley JR, Wiltbank MC, Stevenson JS, Ottobre JS, Garverick HA, Anderson LL. Pregnancy rates per arti®cial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. J Dairy Sci 1997;80:295±300. [17] Guilbault LA, Roy GL, Grasso F, Matton P. In¯uence of pregnancy on the onset of estrus and luteal function after prostaglandin-induced luteolysis in cattle. J Reprod Fertil 1988;84:461±8. [18] Kahn W. Veterinary reproductive ultrasonography. Hannover: Schlutersche Verlagsanstalt und Druckerei GmbH & Co., 1994. p. 83±210. [19] Hanzen C. The role of prostaglandins in human and animal reproductive physiology. J Gynecol Obstet Biol Reprod 1984;13:351±61. [20] Bartolome JA, Archbald LF, Morresey P, Hernandez J, Tran T, Kelbert D, et al. Comparison of synchronization of ovulation and induction of estrus as therapeutic strategies for bovine ovarian cysts in the dairy cow. Theriogenology 2000;53:815±25. [21] Gallo L, Carnier P, Cassandro M, Mantovani R, Bailoni L, Contiero B, et al. Change in body condition score of Holstein cows as affected by parity and mature equivalent milk yield. J Dairy Sci 1996;79: 1009±15. [22] Nanda AS, Ward WR, Williams PCW, Dobson H. Retrospective analysis of the ef®cacy of different hormone treatments of cystic ovarian disease in cattle. Vet Rec 1988;122:155±8. [23] Braun NS, Heath E, Chenault JR, Shanks RD, Hixon JE. Effects of prostaglandin F2 alpha on degranulation of bovine luteal cells on days 4 and 12 of the estrous cycle. Am J Vet Res 1988;49:516±9. [24] Levy N, Kobayashi S, Roth Z, Wolfenson D, Miyamoto A, Meidan R. Administration of Prostaglandin F2a during the early bovine luteal phase does not alter the expression of ET-1 and of its type A receptor: a possible cause for corpus luteum refractoriness. Biol Reprod 2000;63:377±82. [25] LoÂpez-BeÂjar M, Fenech M, LoÂpez-Gatius F. Angiogenesis in luteal cysts of follicular origin in superovulated rabbit does. In: Proceedings of the XVI International Symposium on Morphological Sciences. Symposium 9: Aspects of Female Reproduction, Sun City, South Africa, 2001. p. 70±1.
Reproductive performance of dairy cows with ovarian cysts after different GnRH and cloprostenol treatments F. LoÂpez-Gatiusa,*, M. LoÂpez-BeÂjarb a
Departamento de Produccion Animal, Escuela TeÂcnica Superior de IngenierõÂa Agraria, Universidad de Lleida, Avda. Alcalde Rovira Roure 177, 25198 Lleida, Spain b Department of Veterinarian Anatomy and Embryology, Autonomous University of Barcelona, Barcelona, Spain Received 1 October 2001; accepted 14 February 2002
Abstract Cystic ovarian disease is an important cause of reproductive failure and economic loss for the dairy industry. This report describes two consecutive studies. The objective of the ®rst was to evaluate the response of cows with ovarian cysts to two therapeutic treatments. In the second study, we compared the effectiveness of the best treatment established in Study 1 with that of the Ovsynch protocol. For Study 1, cows were considered to have an ovarian cyst if it was possible to observe a single follicular structure with a follicular antrum diameter 25 mm in the absence of a corpus luteum in three ultrasonographic examinations performed at 7 days intervals. At diagnosis (Day 0), cows were assigned to one of two treatment groups. Cows in Group GnRH/CLP (n 31) were treated with 100 mg GnRH i.m. and 500 mg cloprostenol (CLP) i.m. on Day 14. Cows in Group GnRH±CLP/CLP (n 32) were treated with 100 mg GnRH i.m. plus 500 mg CLP i.m. on Day 0, and 500 mg CLP i.m. on Day 14. The animals were inseminated at observed estrus and monitored weekly by ultrasonography for 4 weeks or until AI. Cows in the GnRH±CLP/CLP group showed a lower cystic persistence rate (15:6% < 45:2%; P 0:01); a higher estrus detection rate (84:4% > 41:9%; P < 0:0001); a higher ovulation rate (75% versus 32.3%; P < 0:0001) and a higher early response rate (31% > 3%; P 0:02) than those in the GnRH/CLP group. For the second study, 128 cows with ovarian cysts were randomly assigned to one of two treatment groups: cows in Group Ovsynch (n 64) were treated with 100 mg GnRH i.m. on Day 0, 500 mg CLP on Day 7, and 100 mm GnRH i.m. 36 h later. Cows in this group were inseminated 24 h after the second GnRH dose (Ovsynch protocol). Cows in Group GnRH±CLP/CLP/GnRH (n 64) were treated as those in the GnRH±CLP/CLP group of Study 1 but received GnRH 32 h after the second CLP treatment and were inseminated 24 h after this. A further group of cows without ovarian cysts inseminated at natural estrus served as the Group Control (n 64). Cows in the GnRH±CLP/CLP/ GnRH group showed a lower cystic persistence rate (10:9% < 46:9%; P < 0:0001); higher ovulation * Corresponding author. Tel.: 34-973-702-500; fax: 34-973-238-264. E-mail address: [email protected] (F. LoÂpez-Gatius).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 9 5 2 - 4
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rate (79:7% > 17:2%; P < 0:0001); higher return to estrus rate (34:3% > 12:5%; P < 0:01) and higher pregnancy rate (28:1% > 3:1%; P < 0:01) than those in Ovsynch; and a similar pregnancy rate (28.1% versus 35.9%) to Control cows. These ®ndings indicate that lactating cows with ovarian cysts can be successfully synchronized and time inseminated using a protocol that combines GnRH and CLP, starting treatment by simultaneously administering both products. This protocol also allows the insemination of cows showing estrus within the ®rst week of treatment. Ovarian cysts were less responsive when treatment was started with GnRH alone. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Dairy cows; Ovarian cysts; GnRH; Cloprostenol
1. Introduction Ovarian cysts are anovulatory follicle-like structures >2.5 cm that persist for 10 days or more in the absence of a corpus luteum [1]. Cystic ovarian disease is a common ovarian disorder diagnosed in dairy cattle and is an important cause of reproductive failure and economic loss for the dairy industry. Depending on plasma progesterone concentration, ovarian cysts can be classi®ed as follicular (low progesterone, 30±85%) or luteal (high progesterone, 15±70%) [2]. Follicular cysts usually have a thin wall and are ¯uctuant on palpation, whereas luteal cysts have a thick wall with luteal tissue and are hard when palpated [3]. The highest incidence rates of cysts have been reported for the intervals before 60 days in milk [4] and between 14 and 40 days postpartum [5]. Follicular cysts, more frequent than luteal cysts during the early postpartum period, progress to become luteal cysts through the luteinization process and ®nally regress [6]. In fact, cysts that develop during the early postpartum period regress spontaneously in more than 50% of cows before insemination programs commence [7]. However, since there is a chance of persistence of cysts beyond 60 days postpartum it may be better to start the treatment immediately rather than waiting for spontaneous recovery [8]. Since the 1970s, hCG and GnRH analogues have been used to treat ovarian cysts. Both hCG and GnRH appear to be equally effective with respect to treatment response and posttreatment fertility [7]. Concerning administration of GnRH, within 28±30 days after a GnRH treatment, 65±80% of cows with ovarian cysts re-establish ovarian cycles [9]. Cysts respond to GnRH by luteinization of the cystic structure [10]. However, luteal function is not always re-established after treatment of follicular cysts with GnRH [11]; Sprecher et al. reported that luteal cysts failed to respond to GnRH during 10 days of treatment [12]. Another option for treatment of cystic ovaries is prostaglandin F2a and its synthetic analogues. Prostaglandins act faster than GnRH: within 2±5 days in over 90% of cases they cause lysis of luteal cysts, either luteinized spontaneously or following GnRH treatment, resulting in normal estrus and fertility [11]. Nevertheless, the response to prostaglandins shown by cows with follicular cysts is poor [13]. Thus, the nature of the cyst needs to be determined before appropriate treatment may be prescribed. The response of cows with ovarian cysts to speci®c GnRH or prostaglandin therapy assigned by a milk progesterone assay has been demonstrated to be much better than treatment with GnRH alone (72% versus 52%) [12]. However, milk progesterone tests are not extensively available, and it is often dif®cult to differentiate luteal from follicular cysts under farm conditions. It is for this
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reason that GnRH is widely used to treat both types of cyst, and prostaglandins used to shorten the interval from GnRH treatment to estrus. Thus, treatment of ovarian cysts with GnRH and a luteolytic agent 9±14 days later, is considered an ef®cient method of cyst recovery without the need to distinguish the two types of ovarian cyst [1,2,7,9]. If all cysts are simultaneously treated with both GnRH and prostaglandins, it would not matter whether the cyst was luteal or follicular, and the interval from treatment to estrus, at least in luteal cysts, would be reduced. In an attempt to treat cystic ovaries using a combination of GnRH and cloprostenol (CLP), combined treatment appeared to have no advantage over the use of GnRH alone, however, prostaglandin was not administered after treatment to shorten the time to estrus [14]. The success of cyst therapy depends mostly on an ef®cient detection of estrus. If estrus is not detected, treated cows cannot be inseminated. Thus, a therapeutic protocol for timedinsemination in cows with ovarian cysts would be of great interest. In a recent study [15], it was suggested that synchronization of ovulation and timed-insemination using the Ovsynch protocol [16] may be an effective method of establishing pregnancy in lactating dairy cows with ovarian cysts. On these grounds, we undertook two consecutive studies on cows which had completed the voluntary waiting period (about 60 days postpartum). The objective of the ®rst study was to evaluate the response shown by cows with ovarian cysts to two treatments: GnRH on Day 0 and CLP administered 14 days later; or simultaneous treatment with GnRH and CLP on Day 0, followed by CLP on Day 14. The second study was designed to compare the reproductive performance after timed AI of cows with ovarian cysts that were treated with GnRH and CLP on Day 0, CLP on Day 14 and GnRH 36 h later, with that shown by animals treated with GnRH on Day 0, CLP on Day 7 and GnRH 36 h later (Ovsynch protocol). 2. Materials and methods 2.1. Study 1 2.1.1. Animals This study was performed on a commercial dairy herd of 650 mature cows in northeastern Spain over the period July 1998 to June 1999. The voluntary waiting period from calving to ®rst AI established for this dairy herd was 60 days. The study population was formed by 63 cows in their ®rst or second lactation period. Mean daily milk production of these cows 10 days before inclusion in the study was 48 kg. The cows were milked three times daily and kept in open stalls. Cows with clinical disorders detected on postpartum examination or during the study were withdrawn from the program. Excluding disorders were: mastitis, lameness, digestive disorders, abnormal genital discharges and pathological abnormalities of the reproductive tract detectable on palpation per rectum. The herd was maintained on a weekly reproductive health program. The reproductive tract of each animal was examined by palpation per rectum within 29±35 days postpartum to check for normal uterine involution and ovarian structures. For ultrasonographic examination, we selected cows with a single follicular structure estimated to be greater than 25 mm, no corpus luteum, and no estrus signs in the previous 14 days. These animals
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were then subjected to three ultrasonographic examinations at intervals of 7 days, starting 14 days after the postpartum examination. A cow was considered to have a ovarian cyst when a follicular structure (of antrum diameter larger than 25 mm) could be observed in all the ultrasonographic examinations in the absence of a corpus luteum (Fig. 1a). 2.1.2. Ultrasonography Cows were diagnosed as having an ovarian cyst by transrectal ultrasonography using a portable B-mode ultrasound scanner (Scanner 100 Vet, equipped with a 5.0 MHz transducer; Pie Medical, Maastricht, The Netherlands). Each ovary was scanned in several planes by moving the transducer along its surface to identify the different structures. The size of cysts was measured using the built in electronic caliper after freezing the image on screen. The largest and the smallest diameters of the follicular antrum were measured and the mean diameter was then recorded. Cows found to have ovarian cysts were subjected to ultrasonographic examinations weekly for 4 weeks or until AI. Ultrasonography was also performed on the ovaries of inseminated cows 11 days after AI. All examinations were performed by the same operator. Cyst regression was registered when a clear decrease of the follicular antrum diameter to a value lower than 20 mm was observed by ultrasound (Fig. 1b). 2.1.3. Treatments and arti®cial insemination At the time of diagnosis of ovarian cysts (Day 0), the cows were alternately assigned to one of two treatment groups (Fig. 2) according to the chronological order of their calving data. Cows in Group GnRH/CLP (n 31) were treated with GnRH (100 mg, i.m.; Dalmarelin, FATRO, Bologna, Italy) on Day 0, and given one luteolytic dose of CLP (500 mg; Estrumate, Schering Plough Animal Health, Madrid, Spain) on Day 14. Cows in Group GnRH±CLP/CLP (n 32) received GnRH plus CLP on Day 0, and CLP on Day 14. All animals were inseminated at observed estrus from Day 0 by the same practitioner using frozen semen from a single ejaculate. 2.1.4. Progesterone analysis Blood samples were taken at the beginning of treatment (Day 0), and 11 days after AI in inseminated cows. All blood was collected from the coccygeal vein into heparinized vacuum tubes. Plasma was separated by centrifugation within 2 h and stored at 20 8C until assayed. Progesterone was determined using solid-phase RIA kits containing antibody-coated tubes, 125 I-labeled progesterone and rabbit antiserum (CS Bio International, Gif-Yvette, France). The RIA method was previously validated for its use in the cow as described by Guilbault et al. [17]. The sensitivity of the assay was 0.05 ng/ml progesterone. Plasma samples showing hormone concentrations below this were assigned the sensitivity value. The intra-assay coef®cient of variation was 16%. Plasma progesterone concentrations were used to classify cysts as luteal (1 ng/ml) or follicular (<1 ng/ml). 2.1.5. Detection of estrus, ovulation and pregnancy diagnosis The animals were inspected for signs of estrus (standing to be mounted) at least four times throughout the day. Cows were inseminated approximately 8±10 h after the ®rst signs of estrus were observed. Ovulation was de®ned as the presence of a corpus luteum and
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Fig. 1. Selected ultrasound pictures of a follicular cyst and its regression from a cow which became pregnant, in the GnRH±CLP/CLP/GnRH group (Study 2): (a) follicular cyst (treatment Day 0); (b) regressed cyst (treatment Day 14); (c) corpus luteum 11 days after AI. All images were from the right ovary. Bar: 20 mm.
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Fig. 2. Schematic summary of treatment groups, ultrasonographic examinations and blood sampling times.
plasma progesterone levels 1 ng/ml 11 days after insemination. A corpus luteum was identi®ed by its size and shape as well as by a granular, gray, oval area, that could be delineated from the remaining ovarian tissue (Fig. 1c) [18]. Pregnancy diagnosis was performed by ultrasonography at 34±40 days postinsemination. 2.1.6. Data analysis The cystic persistence rate was de®ned as the percentage of cows maintaining a follicular structure with an antrum diameter 25 mm in the absence of a corpus luteum on Day 28 after starting treatment. The estrus detection rate was taken as the percentage of cows showing estrus, expressed as a percentage of the total number of cows in each treatment group, between Days 0 (starting treatment) and 28. The ovulation rate was the percentage of inseminated cows that ovulated, expressed as a percentage of the total number of cows in each group at the same period. The early response rate was de®ned as the number of animals that showed estrus and ovulated before ®nishing treatment, as a percentage of the total number of cows in each group between Days 0 (starting treatment) and 14. The pregnancy rate was taken as the number of cows inseminated between Days 0 and 28, that became pregnant, expressed as a percentage of the total number of cows in each group. Treatment regimes were compared in terms of cyst persistence, estrus detection, ovulation, early response and pregnancy rates using the Chi-square test. 2.2. Study 2 2.2.1. Animals, treatments and timed insemination The second study was performed in the same dairy herd over a 14-month-period (1 January 2000 to 28 February 2001). The cows were subjected to the same reproduc-
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tive program and ovarian cysts were diagnosed by ultrasonography as described for Study 1. The study population was formed by 192 cows: 128 cows with ovarian cysts free of other clinical disorders, and 64 healthy animals used as controls. The lactation number was 2.8 and ranged from 1 to 11 lactations. Mean daily milk production 10 days before recruiting for the study was 54 kg. Cows with ovarian cysts were paired by lactation number as ®rst, second, or third or more lactations. One cow from each pair was alternately assigned, according to the chronological order of their calving data, to receive one of two treatments: the Ovsynch program designed by Pursley et al. [16]; or GnRH plus CLP on Day 0 followed by CLP on Day 14, as described in Study 1, plus GnRH 36 h later to synchronize ovulation (Group GnRH±CLP/CLP/GnRH; n 64). Cows in Group Ovsynch (n 64) were treated with GnRH (100 mg i.m.; Dalmarelin) on Day 0 and were given one luteolytic dose of CLP (500 mg i.m.; Estrumate) on Day 7, and 100 mm GnRH i.m. 32 h later. The cows were then inseminated 24 h after the second GnRH treatment in both groups. Thus, including diagnosis and treatment times, cows with ovarian cysts were inseminated between 74 and 80 days after parturition. Ovulation was con®rmed by ultrasonography 11 days after insemination by the presence of a corpus luteum. To assess their subsequent fertility, cows with ovarian cysts were paired with healthy animals (Group Control; n 64) of the same lactational period, inseminated at natural estrus during the same postpartum interval. All animals were inseminated by the same practitioner with frozen semen from a single bull of proven fertility. Cows with ovarian cysts were inseminated without visual detection of estrus, and control cows were inseminated approximately 8±10 h after the ®rst signs of estrus were observed. 2.2.2. Collection of data after insemination Cows returning to estrus from Days 8 to 30 postinsemination were reinseminated. Cows that exhibited estrus after this interval and before pregnancy diagnosis were not inseminated. Pregnancy was diagnosed by ultrasonography at 42 days (Ovsynch group) and at 35 days (GnRH±CLP/CLP/GnRH group) postinsemination. Thus, pregnancy diagnosis was performed 52 days after starting treatment for both groups. Non-pregnant cows with a follicular structure of antrum diameter larger than 25 mm that lacked a corpus luteum, were subjected to a further ultrasonographic examination the following week to determine the presence of an ovarian cyst. 2.2.3. Data analysis The effect of treatment was evaluated in terms of ovulation rate (number of cows with at least one corpus luteum on Day 11 after AI as a percentage of the total number of cows in each group), pregnancy rate (number of pregnant cows after ®rst AI as a percentage of the total number of cows in each group), cows returning to estrus (number of cows returning to estrus 8±30 days postinsemination), and cystic persistence rate (number of cows with an ovarian cyst 7 days after pregnancy diagnosis as a percentage of the total number of cows in each group). Treatment regimes were compared using the Chi-square test. Values corresponding to both studies are expressed as the mean S:D:
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3. Results Throughout both studies, no short cycles or episodes of nymphomania were registered. Selected pictures of a follicular cyst and its evolution from a cow which became pregnant, in the GnRH±CLP/CLP/GnRH group (Study 2), are shown in Fig. 1. 3.1. Study 1 The mean number of days from calving to diagnosis of ovarian cysts (Day 0) was 59:2 1:9 days and ranged from 57 to 63 days. Twenty-six cows (41.3%) had luteal and 37 (58.7%) follicular cysts (plasma progesterone concentration 1 and <1, respectively). Table 1 shows the distribution of the type of cyst per treatment. In the GnRH/CLP group, of 19 cows with previous follicular cysts, 1 cow showed estrus signs within the ®rst week after GnRH treatment, and 9 cows showed estrus within the ®rst week after CLP treatment. Three cows became pregnant. Cystic structures regressed in 1 cow by Day 7 and in 10 cows by Day 14. One cow showing cystic regression on Day 14 developed a new cyst before Day 28. Of the 12 cows with previous luteal cysts, no regression of cystic structures was registered on Day 7 and cysts regressed in 5 cows by Day 14. These 5 cows showed no signs of estrus after CLP treatment. Three of the remaining 9 cows with luteal cysts showed estrus following CLP treatment. One of these 3 cows became pregnant. In the GnRH±CLP/CLP group, three cows with previous follicular cysts and seven with previous luteal cysts showed estrus signs within the ®rst week of GnRH±CLP treatment. Three of these cows became pregnant. Ten cows with previous follicular cysts and seven cows with previous luteal cysts showed estrus during the ®rst week after the second CLP treatment. Six of these cows became pregnant. Of the 18 cows with previous follicular cysts, cystic structures regressed in 3 cows by Day 7 and in 11 cows by Day 14. Of the Table 1 Effect of treatment with GnRH/CLP or GnRH±CLP/CLP 28 days after a diagnosis of ovarian cysts1 Treatment group
n
Type of cyst
Cystic persistence (%)
Estrus detection (%)
Ovulation (%)
Early response (%)
Pregnancy (%)
GnRH/CLP2
19 12 31
Follicular Luteal Total
11/19 (58)a 3/12 (25) 14/31 (45)b
10/19 (53) 3/12 (25)e 13/31 (42)f
7/19 (37) 3/12 (25)I 10/31 (32)j
1/19 (5) 0/12 (0) 1/31 (3)m
3/19 (16) 1/12 (8) 4/31 (13)
GnRH±CLP/CLP3
18 14 32
Follicular Luteal Total
4/18 (22)c 1/14 (7) 5/32 (16)d
13/18 (72) 11/18 (61) 14/14 (100)g 13/14 (93)k 27/32 (84)h 24/32 (75)l
3/18 (17) 4/18 (22) 7/14 (50) 5/14 (36) 10/32 (31)n 9/32 (28)
Different superscripts within the same column denote signi®cant differences detected by the Chi-square test (a, c: P 0:03; m, n: P 0:02; b, d: P 0:01; e±l: P < 0:0001). 1 Results were determined in terms of cystic persistence, estrus, ovulation, early response and pregnancy rates. Percentages were obtained from total number of animals (n) in each group. 2 GnRH/CLP 100 mg GnRH i.m. on Day 0, and 500 mg CLP i.m. on Day 14. 3 GnRH±CLP/CLP 100 mg GnRH i.m. plus 500 mg CLP i.m. on Day 0, and 500 mg CLP i.m. on Day 14.
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Table 2 Effect of treatment regimen on ovulation rate, percentage of cows returning to estrus, pregnancy rate and percentage of non-pregnant cows with ovarian cysts 7 days after pregnancy diagnosis Effects
Control (n 64)
Ovsynch1 (n 64)
GnRH±CLP/CLP/GnRH2 (n 64)
Ovulation rate (%) Returns to estrus (%) Pregnancy rate (%) Cystic persistence (%)
± 21/64 (33)c 23/64 (36)f ±
11/64 (17)a 8/64 (13)d 2/64 (3)g 30/64 (47)i
51/64 (80)b 22/64 (34)ce 18/64 (28)fh 7/64 (11)j
Different superscripts within the same row denote signi®cant differences detected by the Chi-square test (c, d, ce, g, fh: P < 0:01; a, b, f, g, i, j: P < 0:0001). 1 Group Ovsynch 100 mg GnRH i.m. on Day 0; 500 mg CLP i.m. on Day 7 followed by 100 mm GnRH i.m. 32 h later; AI 24 h after the second GnRH treatment. 2 Group GnRH±CLP/CLP/GnRH 100 mg GnRH i.m. plus 500 mg CLP i.m. on Day 0, 500 mg CLP i.m. on Day 14 followed by 100 mm GnRH i.m. 32 h later; AI 24 h after the second GnRH treatment.
14 cows with luteal cysts, cystic structures regressed in 10 cows by Day 7 and in 4 cows by Day 14. Ovulation was con®rmed 11 days after AI by the presence of a corpus luteum and luteal activity in 10 of 13 inseminated cows in the GnRH/CLP group, and in 24 of 27 inseminated cows in the GnRH±CLP/CLP group. Four of the cows that failed to ovulate developed cysts once again and were diagnosed as having persistent cysts: two cows with previous follicular cysts in the GnRH/CLP group, and one cow with previous follicular and another with previous luteal cysts in the GnRH±CLP/CLP group. All 11 cows showing estrus within the ®rst week after the onset of treatment ovulated. Table 1 shows the persistent cyst, estrus detection, ovulation, early response and pregnancy rates for cows in the two treatment groups. A signi®cant effect by treatment was shown on persistent cyst, estrus detection, ovulation and early response rates. Pregnancy rates were not signi®cantly affected. 3.2. Study 2 The mean number of days from calving to diagnosis of ovarian cysts (Day 0) was 60:5 2:1 days, ranging from 57 to 63 days. A signi®cant effect by treatment was shown on the ovulation, return to estrus and pregnancy rates, and on the percentage of open cows with ovarian cysts 7 days after pregnancy diagnosis (Table 2). 4. Discussion Study 1 shows the effectiveness of GnRH plus CLP and CLP administered 14 days later in resolving cysts without the need for diagnosing the type of ovarian cyst. The cystic persistence rate (16%) was signi®cantly lower than that observed using GnRH alone plus CLP 14 days later (45%). Furthermore, estrus detection and ovulation rates were signi®cantly higher in response to GnRH±CLP/CLP treatment. In this last treatment
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group, 10 cows (31%) showed estrus and ovulated within the ®rst week after treatment onset, compared to 1 cow (3%) in the GnRH/CLP group. This ®nding is of considerable interest for dairy farms with an ef®cient estrous detection system: it is suggested that using the GnRH±CLP/CLP protocol, a proportion of cows could be inseminated before ®nishing treatment, thus reducing the number of open days. Follicular cysts showed a worse recovery response in the GnRH/CLP than in the GnRH± CLP/CLP group (42% versus 78% regression, respectively). These results indicate a possible synergistic effect of CLP on GnRH in luteinizing follicular cysts. Exogenous prostaglandins can induce a hypophysial LH release [19]. Conversely, GnRH does not appear to inhibit the luteolytic effect of CLP on cows with luteal cysts, as previously reported [14]. Seven of 14 cows with luteal cysts in the GnRH±CLP/CLP group showed estrus and ovulated within the ®rst week after GnRH±CLP treatment. However, in the GnRH/CLP group, no cows with luteal cysts showed estrus before Day 14. A poor response or lack of response of luteal cysts to GnRH has also been previously reported [11,12]. No cystic regression was registered by ultrasonography on Day 7 of treatment in cows with luteal cysts, and 5 of 12 cows, in which cysts regressed 14 days after GnRH treatment, were not responsive to subsequent CLP. It is likely that the effect of prostaglandins on luteal cysts following GnRH treatment could be improved by reducing the interval from GnRH to prostaglandin treatment. Our Study 1 results differ in part from those of Dinsmore et al. [14], in which simultaneous administration of GnRH and CLP appeared to have no advantage over GnRH alone. In our study, the administration of CLP 14 days after treatment probably had the effect of inducing estrus in more cows, leading to differences between treatments. The present study was performed on a single dairy herd, eliminating the herd effect, to ensure that animals receiving different treatment regimes were subjected to the same management practices. Dinsmore et al. [14] performed their study on 75 cows from 32 herds, a factor that may have masked ovarian cystic responses to treatments. Based on the ®ndings of Study 1, the GnRH±CLP/CLP treatment was used in the second study which involved the administration of GnRH 36 h after the second CLP treatment, to synchronize ovulation. We compared the reproductive performance of lactating dairy cows subjected to the Ovsynch protocol (Ovsynch group) with that of cows simultaneously administered GnRH and CLP on Day 0, CLP 14 days later and GnRH 36 h after this (GnRH±CLP/CLP/GnRH group), followed by timed insemination. Best results were associated with GnRH±CLP/CLP/GnRH treatment, with a higher ovulation rate (80%), return to estrus rate (34%) and pregnancy rate (28%) recorded, along with a lower cystic persistence rate (11%) 52 days after starting treatment. For cows in this group, similar return to estrus and pregnancy rates were recorded after timed insemination for noncystic (33 and 36%, respectively) and cystic cows (34 and 28%, respectively). While this work was in progress, a paper by Bartolome et al. was published [20], in which the authors reported that using the Ovsynch protocol as a therapeutic strategy for bovine ovarian cysts in dairy cows, similar pregnancy rates were obtained after timed insemination in cows with (23.6%) and without (31.5%) cysts. Our results are in clear disagreement: only 2 out of 64 cows with cysts (3%) became pregnant following the Ovsynch protocol plus timed insemination. The explanation for this discrepancy could be related to the variable ``number of days in milk''. In the study by Bartolome et al. [20], days in milk ranged from 65 to 533 days
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(over 75% of cows ranged from 133 to 533 days), a period of positive energy balance for most cows [21]. Our study was conducted during a short postpartum period; ovarian cysts were diagnosed within 57±63 days after calving (a common voluntary waiting period from calving to ®rst insemination). During this period, the metabolic demands of lactation often hinder ®nal follicular maturation and ovulation. It is also possible that cows with ovarian cysts were recovering from extended periods of negative energy balance after calving. The predominance of follicular cysts registered in Study 1 (59% follicular versus 41% luteal) could be accounted for by the metabolic status of the cows. Ovarian cysts are dynamic in nature, and cysts classi®ed as follicular may be later classi®ed as luteal during their lifespan [1,7]. From a clinical perspective, we might consider follicular cysts as a ®rst stage in the ovarian cystic condition, and luteal cysts as a sign of recovery. With these points in mind, we may propose that when cows enter the positive balance period 90±120 postpartum, as the number of days in milk increases, the probability of a predominance of luteal cysts also rises. If so, the Ovsynch protocol would be more ef®cient in advanced lactation periods in which luteal cysts predominate. As noted above in Study 1, prostaglandins should be more ef®cient if applied before Day 14 after GnRH treatment in cows with luteal cysts. On the contrary, when prostaglandins are administered on Day 7 (Ovsynch protocol), follicular cysts have too short a period for recovery. Nanda et al. [22] demonstrated that luteinized follicular cysts showed a poor response to prostaglandins administered 7 days after GnRH treatment. Luteal tissue producing progesterone for less than 7 days after GnRH treatment in cows with follicular cysts may be slightly responsive to prostaglandins, perhaps as occurs in the early luteal period of cyclic cows [23]. Corpus luteum refractoriness to prostaglandin F2a during the early bovine luteal phase has been partly related to the fact that neovascularization is as yet incomplete [24]. We recently demonstrated that neovascularization in the luteinized area of cysts in rabbits occurs only at advanced stages of cystic luteinization, with endothelial cells remaining near the basament membrane area of the cysts during the early cystic luteinization process [25]. In conclusion, the present ®ndings indicate that lactating cows with ovarian cysts can be successfully synchronized and time inseminated using a protocol that combines GnRH and CLP, starting treatment by the simultaneous administration of both products. This protocol also allows cows showing estrus within the ®rst week of treatment to be inseminated in farms with an ef®cient estrus detection system. In contrast, ovarian cysts are less responsive to starting treatment with GnRH alone. Acknowledgements The authors thank Ana Burton for assistance with the English translation. This study received ®nancial support from the CTT of University of Lleida (C-0243 with Schering Plough Animal Health, Spain). References [1] Garverick HA. Ovarian follicular cysts in dairy cows. J Dairy Sci 1997;80:995±1004. [2] Farin PW, Estill CT. Infertility due to abnormalities of the ovaries in cattle. Food Anim Pract 1993;9:291±308.
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