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Persistent ovarian follicles in dairy cows: a therapeutic approach
ELSEVIER
PERSISTENT OVARIAN FOLLICLES IN DAIRY COWS: A THERAPEUTIC APPROACH F. Lopez-Gatius, la P. Santolaria,2 J. Yaniz, ’ J. Ruthant
and M. Lopez-Bejar
‘Animal Production, University of Lleida; 2Animal Production, University of Zaragoza 3Veterinarian Anatomy and Embryology, Autonomous University of Barcelona, Spain Received for publication: Septenker 21 , 2000 Accepted: April 9, 2001 ABSTRACT Anestrus is common during the postpartum period in high-producing dairy cows. In a previous investigation, we were able to diagnose persistent follicles of 8 to 12 mm in anestrous cows. This report describes 2 consecutive studies. The objectives of the first were to 1) assess the association of persistent follicles with anestrus; and 2) evaluate 2 therapeutic treatments. In the second study, we compared the effectiveness of the best treatment established in Study 1 with the Ovsynch protocol. For Study 1, anestrous cowswere considered to have a persistent follicle if it was possible to observe a single follicular structure > 8 mm in the absence of a corpus luteum or a cyst in 2 ultrasonographic examinations performed at an interval of 7 d. At diagnosis (Day 0), cows were assigned to 1 of 3 treatment groups. Cows in Group GnRH/PGF (n= 17) were treated with 100 ug GnRH im, and 25 mg PGFh im on Day 14. Cows in Group PRID (n=lS) were fitted with a progesterone releasing intravaginal device (PRID, containing 1.55 g of progesterone) for 9 d and were given 100 ng GnRH im at the time of PRID insertion, and 25 mg PGFh im on Day 7. Cows in Group Control (n=lS) received no treatment. The animals were inseminated at observed esnus and were monitored weekly by ultrasonogmphy until AI or 5 weeks from diagnosis. Blood samples were also collected on a weekly basis for progesterone determination. The mean size of persistent follicles on Day 0 was 9.4 + 0.04 mm. Progesterone levels were < 0.2 ng/mL during the first 35 d in 16 of 18 Control cows. Cows in the PRID group showed a lower persistent follicle rate (16.7% < 70.6% < 88.9%; P < 0.0001; PRID vs GnRH/PGF vs Control, respectively); a higher estms detection rate (83.3% > 29.4% > 11.l%; P < 0.0001) and a higher pregnancy rate (27.8% > 59% > 0%; P = 0.02). For the second study, 145 cows with persistent follicles were randomly assigned to 1 of 2 treatment groups: cows in Group Ovsynch (n=73) were treated with 100 lrg GnRH im on Day 0,25 mg PGFh im on Day 7, and 100 pm GnRH im 32 h later. Cows in this group were inseminated 16 to 20 h after the second GnRH dose (Ovsynch protocol). Cows in Group PRID (n=72) were treated as those in the PRID group of Study 1, and were inseminated 56 h after PRID removal. Cows in the PRID group showed a higher ovulation rate (84.8% > 8.2%; P < 0.0001); a higher pregnancy rate (34.2% > 4.1%; P < 0.0001) and lower follicular persistence rate (22.2% < 63%; P < 0.0001) than those in Ovsynch. Our results indicate that persistent follicles affect cyclic ovarian function in lactating dairy cows. Cows with persistent follicles can be successfully synchronized and time inseminated using progesterone, GnRH and PGF2a but show a limited response to treatment with GnRH plus PGF2a. 0 2001 by Elsevier Sc~ence Inc. Key words: dairy cows, ovary, persistent follicles, progesterone, G&II, PGFk Acknowledgments The authors thank Ana Burton for assistance with the English translation. aCorrespondence and reprint requests: Escuela T&c&a Superior de Ingenieria Agraria Universidad de Lleida, Avda. Alcalde Rovira Roure 177,25 198 Lleida, Spain, e-mail: [email protected] Theriogenology 56:64M59. 2001 0 2001 Elsevier Science Inc.
0093-691X/01/$-see front matter PII: SOO93-691X(01)00596-9
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INTRODUCTION There are 2 or 3 waves of follicular growth in the normal estrus cycle of cattle. Each wave involves the development of a large dominant follicle and smaller subordinate follicles. When the largest follicle reaches a mean diameter of 8.5 mm, follicular deviation begins (9). The deviation mechanism consists of the reduction or cessation of growth of the remaining follicles (subordinate follicles) while the largest follicle becomes dominant and increases in size. Luteinizing hormone appears to regulate the function of the dominant follicle (23, 26, 28). Final maturation and ovulation of the dominant follicle is associated with a rapid LH pulse frequency and follicular atresia is associated with a slow frequency (28). However, low progesterone concentrations or treatment with synthetic progestogens used to synchronize estrus in beef cattle have been associated with intermediate LH pulse frequencies (1 pulse per 1 to 2 h), maintaining estradiol production by a dominant follicle which becomes persistent (24, 28, 29). Persistence of the dominant follicles reduces fertility (2, 25, 28), and acute progesterone administration regresses induced persistent follicles, improving subsequent conception rates (1, 17). Follicles dominant for an extended period of time were called “prolonged“ or “persistent” dominant follicles (27), and the term “persistent follicles” is used extensively in beef cattle (13). Induced persistent follicles are smaller than typical ovarian cysts (5), and may be considered a cause of anestrus (19). Anestrus, a common state during the postpartum period in high-producing dairy cows, has major economic implications since it increases the length of the calving interval. Anestrus is either a failure to exhibit estrus behavior in cyclic cows - the most frequent finding in cows with luteinized cysts - or is due to inactive ovaries. Lactating dairy cows are diagnosed as having inactive ovaries when they show no behavioral signs of estrus, accompanied by failure to detect a corpus luteum or cyst at 2 consecutive examinations per rectum performed at an interval of 7 d (16). In a previous study (15), 13% of cows were diagnosed as having inactive ovaries (range 12 to 14.2% in different farms). These animals were then subjected to transrectal ultrasonography on two more occasions (7 d apart) after the rectal examinations. One or 2 follicles of 8 to 12 mm were detected in all animals during the first ultrasonography. Similar structures persisted in each ovary 7 d later and were considered persistent ovarian follicles and thus a possible cause of anestrus. As far as we are aware, this is the only report of clinical cases of spontaneous persistent ovarian follicles in lactating dairy cows, and no therapeutic approaches for this condition have been described in the literature. On these grounds, we undertook two consecutive studies. The objectives of the first study were to 1) assess the association of persistent follicles with anestrus; and 2) evaluate the 2 treatments: GnRH plus PGF2cr administered 14 d later; and progesterone for 9 d, with GnRH given at the start of treatment and PGF2a given on Day 7. The second study was designed to compare reproductive performance after timed AI of cows with persistent follicles that were treated with GnRH, progesterone, and PGF2a as in Study 1 with that of animals treated with GnRH, PGF2a on Day 7 and GnRH 32 h later (Ovsynch protocol). MATERIALS AND METHODS Study 1 All drugs were purchased from Sanoti Salud Animal (Barcelona, Spain) unless otherwise indicated.
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Animals. This study was performed from September 1998 to May 1999 on a commercial dairy herd in northeastern Spain. Mean annual milk production of the herd for this period was 9840 kg per cow. The cows were milked 3 times daily and were kept in open stalls. The animals were grouped according to production and age such that one group was formed by cows in their first lactation period. The study was performed on cows from this group. Cows with clinical disorders detected on postpartum examination or during the study were removed from the program. The disorders included mastitis, lameness, digestive disorders, abnormal genital discharges and pathological abnormalities of the reproductive tract detectable on palpation per rectum. Thus, the study population was formed by 53 cows in their first lactation period. The herd was maintained on a weekly reproductive health program. The reproductive tract of each animal was examined by palpation per rectum within 43 to 49 d postpartum to check for normal uterine involution and ovarian structures. For ultrasonographic examination, we selected cows with a single follicular structure of 8 to 15 mm, no corpus luteum or cyst, and no estrus signs in 7 d. These animals were then subjected to 2 ultrasonographic examinations at an interval of 7 d. A cow was considered to have a persistent follicle when the follicular structure detected by rectal palpation could be observed in both ultrasonographic examinations in the absence of a corpus luteum or cyst. Ultrasonography. Cows were diagnosed as having a persistent follicle 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 follicles was measured using the in-built electronic caliper after freezing the image on screen. The largest and the smallest diameters were measured and the mean diameter was then recorded. Cows were submitted to ultrasonographic examinations at 7 d intervals until AI or 5 weeks of diagnosis. The ovaries of inseminated cows were also examined ultrasonographically 10 d after Al. All examinations were performed by the same operator. Treatments and artilicial insemination. At the time of diagnosis of persistent follicles (Day 0), the cows were alternately assigned to 1 of 3 treatment groups (Figure 1) according to the chronological order of their calving data. Cows in Group GnRH/PGF (n= 17) were treated with GnRH (100 pg, im; Cystorelyn) on Day 0, and given one luteolytic dose of PGFla (25 mg im; Enzaprost) intravaginal 9 d without the time of received no
on Day 14. Cows in Group PRID (n=18) received a progesterone releasing device (PRID, containing 1.55 g of progesterone) on Day 0 that was maintained for the estradiol benzoate capsule. These animals were also given 100 ug GnRH im at PRID insertion and 25 mg PGF za im on Day 7. Cows in Group Control (n=l8) treatment.
All animals were inseminated semen from a single ejaculate.
at observed estrus by the same practitioner
using frozen
Progesterone analysis. Blood samples were taken just before the second (Day 0) and at all the following ultrasonographic examinations. A blood sample was also obtained IO d after AI in inseminated cows, All blood was collected into heparinized vacuum tubes from the coccygeal vein. Plasma was separated by centrifugation within 2 h and then was stored at -20°C until assayed. Progesterone was measured using solid-phase RIA kits containing antibody-coated
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tubes, ‘251-labeled progesterone and rabbit antiserum (CS Bio International, Gif-Yvette, France). The RIA method was previously validated as described for the cow by Guilbault et al. (11). 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 coefficient of variation was 9.6%. Plasma progesterone concentrations were used to classify cows as showing (2 lng/mL) or not showing (< lng/mL) luteal activity.
Group GnRHlPGF Day 14 Day 0 ()______ _____________________________~_______-___o GnRH PGF2,
AI at estrus
Group PRID Day 0 0 PRID insertion + GnRH
Day 7 1 PGP2,
Day 9 ______________________ 0 PRlD removal AI at es&us
Group Control Day 0 0 _____________________--_________________________
AI at%trus
Ultrasonographic examinations and blood sampling times Day 7... At 7 d intervals until AI... Day 0 ~_____________-____-+--_____________________-________ Figure 1. Schematic summary of treatment groups, ultrasonographic sampling times.
10 d after AI
examinations
and blood
Detection of estrus and pregnancy diagnosis. The animals were inspected for signs of estrus (standing to be mounted) at least 4 times throughout the day. Cows were inseminated approximately 8 to 10 h after the first signs of estrus were observed. Pregnancy diagnosis was performed by palpation per rectum at 34 to 40 d postinsemination. Data analysis. The follicular persistence rate was defined as the percentage of cows maintaining an 8 to 15 mm follicle on Day 35. The estrus detection rate was taken as the percentage of cows showing estrus between Days 0 and 35. The pregnancy rate was defined as the number of cows that became pregnant expressed as a percentage of the total number of cows in each group at the same period.
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Treatment regimes were compared in terms of follicular persistence, estrus detection and pregnancy rates using the Chi-square test. Data from Control cows were used to determine the possible association of follicular persistence with anestrus. Study 2 Animals, treatments and timed insemination. The second study was performed in another dairy farm, less than 30 km away from the first, over the period May to September 1999. Milking and management practices were similar to those of the previous farm. Mean annual milk production was slightly over 10,000 kg per cow. The cows were subjected to the same reproductive program and follicular persistence was diagnosed as described for Study 1. The study population was formed by 145 cows with persistent follicles that were free of other clinical disorders. This time, the lactation number was 1.8 and ranged from 1 to 3 lactations. Cows were paired by lactation number and 1 cow from each pair was randomly assigned to receive one of two treatments: the Ovsynch program designed by Pursley et al. (21); and progesterone, GnRH and PGF2a as described in Study 1. Cows in Group Ovsynch (n=73) were treated with GnRH (100 pg im; Cystorelyn) on Day 0 and were given one luteolytic dose of PGF2, (25 mg im; Enzaprost) on Day 7, and 100 pm GnRH im 32 h later. The cows were then inseminated 16 to 20 h after the second GnRH injection. Cows in Group PRID (n=72) were treated as those in the PRID group of Study 1 and were inseminated 56 h after PRID removal. All animals were inseminated by the same practitioner with frozen semen from a single ejaculate without visual detection of estrus. Two cows lost their PRID device and were excluded from the study. Collection of data after insemination. The ovaries of inseminated cows were examined ultrasonographically 10 d after AI. 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 34 to 40 d postinsemination. Open cows with a corpus luteum were included in a synchronization program. Open cows with a single follicle larger than 8 mm with no corpus luteum or cyst were subjected to a further ultrasonographic examination the next week to determine the presence of a persistent follicle. Data analysis. The effect of treatment was evaluated in terms of the dependent variables defined in Table 1. The independent variables were the treatment groups.
Table 1. Definition of dependent variables. Variable Definition Number of cows with at least 1 corpus luteum on Day 11 after AI as Ovulation rate a percentage of the total number of cows in each group Number of pregnant cows after first AI as a percentage of the total Pregnancy rate number of cows in each group Cows returning to estrus Number of cows returning to estrus 8 to 30 d postinsemination as a percentage of the total number of cows in each group Follicular persistence Number of cows with a persistent follicle 7 d after pregnancy diagnosis as a percentage of the total number of cows in each group
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Treatment regimes were compared using the Chi-square test. Values corresponding to both studies are expressed as the mean f standard deviation (SD). RESULTS Study 1 The mean number of days from calving to diagnosis of persistent follicles (Day 0) was 61 .S f 2.1 d, ranging from 57 to 63 d. Cows showed no luteal activity on Day 0. Mean plasma progesterone concentration was 0.07 * 0.03 ng/mL, ranging from 0.05 to 0.2 ng/mL. Plasma progesterone concentrations were I 0.2 ng/mL throughout the study for Control cows with persistent follicles. Mean persistent follicle sizes at Day 0 were 9.6 rt 0.05, 9.4 f. 0.06 and 9.3 + 0.06 mm for cows in the GnRH/PGF, PRID and Control groups respectively. All cows showing no signs of estrus had persistent follicles that were of similar size throughout the study. One cow in the GnRH/PGF group showed estrus signs during the first week after GnRH treatment. The remaining 16 cows showed no luteal activity on Day 7. Regression of follicles and luteal activity were registered in 5 cows on Day 14. Follicular regression was defined as the absence of follicles larger than 8 mm. Four of these cows reached estrus during the following week. The cow showing luteal activity which subsequently failed to display signs of estrus developed a new persistent follicle 7 d after prostaglandin treatment. In the remaining 11 cows, follicles persisted throughout the 5 week study period with the exception of one cow in which a new persistent follicle developed in the contralateral ovary, the first having regressed in the third week. Only I cow became pregnant after prostaglandin treatment. The average plasma progesterone concentration in the PRID group was 2.8 f 0.3 ng/mL progesterone, ranging from 1.5 to 4.2 ng/mL, on Day 7 after PRID insertion. Persistent follicles regressed in 15 of the 18 cows during the first week. These 15 cows (83%) showed estrus signs 2 to 4 d after PRID withdrawal: 13115 (72%) cows reached estrus at 48 h, 1 at 60 h and the remaining cow at 96 h. The remaining 3 cows maintained their persistent follicles during the 5 weeks of study. Five cows became pregnant. Only 2 Control group cows showed estrus after diagnosis, on Days 20 and 26 respectively. Persistent follicles were still present in the 16 remaining anestrous cows throughout the 5-week study period. However, one cow developed an additional persistent follicle in the same ovary during the fourth week and both follicles were present on Day 35. A further cow developed a persistent follicle in the contralateral ovary after regression of the first follicle in the fourth week. No cows became pregnant. Table 2 shows follicular persistence, estrus detection and pregnancy rates for cows in the 3 treatment groups. A significant effect was shown by treatment on all the variables.
Theriogenology
Table 2.
Group
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Effect of treatment with GnRH/PGF or PRID on cows 35 days after they had been diagnosed with persistent follicles. Results are measured by follicular persistence, estrus and pregnancy rates.
Estrus detection rate Follicular persistence Pregnancy rate (%)’ (%)h rate (%)a GnRH/PGF 17 29.4 70.6 5.9 PRID 18 16.7 83.3 27.8 Control 18 88.9 11.1 0 GnRH/PGF = 100 ug GnRH im on Day 0, and 25 mg PGFln im on Dayl4. PRID = 1.55 g intravaginal progesterone for 9 d, plus 100 ug GnRH im on Day 0 followed by 25 mg PGFla im on Day 7. Control = untreated cows. a.hProportions were different (P < 0.0001) when compared in a 3x2 contingency table using the Chi-square test. ‘Proportions were different (p = 0.02) when compared in a 3x2 contingency table using the Chi-square test. n
Ovulation was confirmed by the presence of a corpus luteum inseminated cows 10 d after AI. No double ovulations were recorded.
and luteal activity
in all
Study 2 The mean number of days recorded from calving to diagnosis was 60.2 ?_ 1.6 d, ranging from 57 to 63 d.
of persistent
follicles
(Day 0)
A significant effect was shown by treatment on the ovulation rate, pregnancy rate and the percentage of open cows with persistent follicles 7 d after pregnancy diagnosis (Table 3). The proportion of cows returning to estrus was unaltered by treatment.
Table 3. Effect of treatment regimen on ovulation rate, pregnancy rate, percentage of cows returning to estrus and percentage of open cows with persistent follicles 7 d after pregnancy diagnosis. Effects Ovsynch (n=73) PRID (n=72) P Ovulation rate (%) 8.2 84.8
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Three open cows in the Ovsynch group and 2 in the PRID group were found to have cystic ovaries after pregnancy diagnosis. Six open cows in Ovsynch and 1 in PRID had 2 persistent follicles of similar size on Day 7 d after pregnancy diagnosis. Follicular persistence was also recorded in the last 7 cows. DISCUSSION The effects of spontaneous persistent follicles on cyclic ovarian function in lactating dairy cows is documented for the first time in Study 1. Since progesterone levels were stable and very low for 35 d in 16 out of 18 Control cows, we believed that anestrus was most probably associated with persistent follicles. In a previous study including data from 3 dairy farms (15), all anestrous cows diagnosed as having inactive ovaries had persistent follicles with no corpora lutea or cysts. Indeed, an extensive epidemiological review of the incidence of persistent follicles in anestrous cows would be of major interest. The development of persistent follicles in lactating dairy cows may result from a failure to resume ovarian activity in the postpartum period. Selection of the first dominant follicle can occur as early as 7 to 15 d after calving, and 1 to 3 follicular waves can be observed before first ovulation, which usually occurs between 15 and 2.5 d post partum (22). Hereafter the cows resume their cyclic ovarian function. Nevertheless, the length of the postpartum anovulatory period can be affected by a number of factors such as a negative energy balance (4), milk production, age, calving season and reproductive diseases (10, 20). The development of anovulatory or cystic follicles may in these cases prolong the interval until first ovulation to 40 or 50 d postpartum (3). In the present study, although there was no interest in determining the time of first ovulation, our data indicate that cows with persistent follicles can exceed 90 d postpartum before they return to cyclicity. The mean number of days from calving to diagnosis of persistent follicles was 61.8 + 2.1 d, and these persisted for 35 d in 16 of 18 Control cows. It is possible that cows with persistent follicles were recovering from extended periods of negative energy balance after calving. Moreover, the metabolic demands of lactation could also hinder final follicular maturation and ovulation. In Study 1, follicular persistence was characterized by the presence of a follicular structure similar in size to that of a dominant follicle (> 8 mm). This follicle was morphologically dominant for an extended period of time. The main clinical effect of the persistent follicles, anestrus, may be compared with that of luteinized cysts. Three phenomena described for ovarian follicular cysts (7, 12) were also observed in Control cows with persistent follicles: persistence (15 cows), spontaneous recovery (2 cows spontaneously ovulated) and turnover (1 cow developed a new persistent follicle in the contralateral ovary after regression of the first). Replacement by a new follicle in the contralateral ovary was also observed in one cow from the GnRH/PGF group. Due to our work conditions, in which only a single ultrasonographic examination was performed per week, it is possible that more follicles were replaced by new persistent follicles in the same ovary, however, persistence for an extended period appears to be the foremost feature of persistent follicles. In a recent study (19), in which ultrasonography was conducted each day for 26 d and then on alternate days, induced persistent follicles remained detectable up to 52 d. The dynamic nature and turnover of spontaneous persistent ovarian follicles in dairy cows requires further investigation.
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The administration of GnRH and its synthetic analogues acutely increases LH and FSH levels in the peripheral circulation for a 2 to 4 h period (6). In cyclic cows, GnRH induces ovulation or atresia of the dominant follicle, depending on the stage of follicular development and the emergence of a new follicular wave within 3 to 4 d after treatment (30, 3 1). Exogenous GnRH has also been extensively used for ovarian cyst treatment (8). Cysts respond to GnRH by luteinization of the cystic structure and the success rate of treatment is currently around 80% (8, 30). However, in the present study, the administration of GnRH induced ovulation or atresia in only 6 of 17 cows (35%) and no cows showed luteal activity on Day 7 in the GnRH/PGF group (Study 1). Only 6 of 73 cows (8%) ovulated after the second GnRH injection in the Ovsynch group (Study 2). Furthermore, follicular persistence was recorded in 46 of 73 cows (63%) 40 d after Ovsynch treatment (Study 2). These results indicate a poor response of cows with persistent follicles to GnRH. It would appear that factors other than LH surge may induce extended periods of anovulatory activity in the presence of persistent follicles. Ovarian cysts are anovulatory follicle-like structures greater than 2.5 cm that persist for 10 d or more in the absence of a corpus luteum (8). Although smaller in size, persistent follicles would be expected to respond to treatment in the same way as follicular cysts. Thus, two types of treatment approaches adapted from follicular cyst therapy were tested in Study 1. Treatment of follicular cysts with GnRH and with a luteolytic agent 14 d later (GnRH/PGF group), has been shown to be an efficient method for cyst recovery (8, 18). The treatment regime progesterone for 9 d, GnRH on Day 0, and PGF2a on Day 7 (PRID group) has also been proposed as appropriate treatment for cysts (30). Here, best results were associated with PRID treatment, which gave rise to the highest overall estrus detection rates (83%), a high degree of synchrony (72% of cows started estrus after 48 h of PRID removal), and a lower follicular persistence rate and higher pregnancy rate than GnRH/PGF treatment. This treatment protocol permitted timed insemination at 56 h in Study 2. The administration of progesterone and GnRH in cows in the PRID group (Study 1) induced the regression of persistent follicles in most cows (15 of 18) during the first week. These findings agree in part with those reported for beef cattle. In beef cows that developed persistent ovarian follicles after estrus synchronization with progestogens, the acute administration of progesterone decreased LH secretion (17) atresia of persistent follicles and recruitment of a new dominant follicle (1, 17, 28). Progesterone was probably the most efficient component of the PRID protocol used in the present study. In Study 2, we compared the reproductive performance of lactating dairy cows following the Ovsynch protocol (Ovsynch group) with the administration of progesterone, GnRH and PGF2a (PRID group). Best results were also associated with PRID treatment, with a higher ovulation rate (85% after PRID removal) and pregnancy rate (34%) recorded along with a lower follicular persistence rate 40 d after treatment (22%). The pregnancy rate was similar to that of lactating dairy cows in natural estrus (36%) in the geographical area of the study (14). In conclusion, the present findings indicate that lactating dairy cows with persistent follicles can be successfully synchronized and time inseminated using a protocol that combines progesterone, GnRH and PGF2a. In contrast, persistent follicles were scarcely responsive to treatment with GnRH plus PGF2o.
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REFERENCES 1. Anderson LH, Day ML. Acute progesterone administration regresses persistent dominant follicles and improves fertility of cattle in which estrus was synchronized with melengestrol acetate. J Anim Sci 1994;72:2955-2961. 2. Austin EJ, Mihm M, Ryan MP, Williams DH, Roche JF. Effect of duration of dominance of the ovulatory follicle on onset of estrus and fertility in heifers. J Anim Sci 1999;77:22 192226. 3. Beam SW, Butler WR. Energy and ovarian follicle development prior to the first ovulation postpartum in dairy cows receiving three levels of dietary fat. Biol Reprod 1997;56:133142. 4. Beam SW, Butler WR. Effects of energy balance on follicular development and first ovulation in postpartum dairy cows. J Reprod Fertil 1999;Suppl 54:41 l-424. 5. Bigelow KL, Fortune JE. Characteristics of prolonged dominant versus control follicles: follicle cell numbers, steroidogenic capabilities, and messenger ribonucleic acid for steroidogenic enzymes. Biol Reprod 1998;58:1241-1249. 6. Chenault JR, Kratzer DD, Rzepkowski RA, Goodwin MC. LH and FSH response of holstein heifers to fertirelin acetate, gonadorelin and buserelin. Theriogenology 1990;34:81-98. 7. Cook DL, Smith CA, Parfect JR, Youngquist RS, Brown EM, Garverick HA. Fate and turnover rate of ovarian follicular cysts in dairy cattle. J Reprod Fertil 1990;90:37-46. 8. Garverick HA. Ovarian follicular cysts in dairy cows. J Dairy Sci 1997;80:995-1004. 9. Ginther OJ. Selection of the dominant follicle in cattle and horses. Anim Reprod Sci 2000;60-61:61-79. 10. Grohn YT, Rajala-Schultz PJ. Epidemiology of reproductive performance in dairy cows. Anim Reprod Sci 2000;60-61:605-614. 11. Guilbault LA, Roy GL, Grass0 F, Matton P. Influence of pregnancy on the onset of oestrus and luteal function after prostaglandin-induced luteolysis in cattle. J Reprod Fertil 1988; 84:461-468. 12. Hamilton SA, Garverick HA, Keisler DH, Xu ZZ, Loos K, Youngquist RS, Salfen BE. Characterization of ovarian follicular cysts and associated endocrine profiles in dairy cows. Biol Reprod 1995;53:890-898. 13. Kinder JE, Kojima FN, Bergfeld EGM, Wehrman ME, Fike KE. Progestin and estrogen regulation of pulsatile LH release and development of persistent ovarian follicles in cattle. J Anim Sci 1996;74: 1424-1440. 14. Lopez-Gatius F. Reproductive performance of lactating dairy cows treated with cloprostenol, hCG and estradiol benzoate for synchronization of es&us followed by timed AI. Theriogenology 2000;54:551-558. 15. Lopez-Gatius F, Lopez-B&jar M, Rutllant J, Yaniz J, Santolaria P. Persistent follicles in high-producing dairy cows Proc EETA Annual Meeting, Lyon France, 1999; 190 abst. 16. Markusfeld 0. Inactive ovaries in high-yielding dairy cows before service: Aetiology and effect on conception, Vet Ret 1987; 12 1: 149-I 53. 17. McDowell CM, Anderson LH, Kinder JE and Day ML. Duration of treatment with progesterone and regression of persistent ovarian follicles in cattle. J Anim Sci 1998;76: 850-855. 18. Nanda AS, Ward WR, Williams PCW, Dobson H. Retrospective analysis of the efficacy of different hormone treatments of cystic ovarian disease in cattle. Vet Ret 1988;122: 155-l 58.
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19. Noble KM, Tebble JE, Harvey D, Dobson H. Ultrasonography and hormone profiles of persistent ovarian follicles (cysts) induced with low doses of progesterone in cattle. J Reprod Fertil 2000;120:361-366. 20. Opsomer G, Griihn YT, Hertl J, Coryn M, Deluyker H, de Kruif A. Risk factors for post pat-turn ovarian dysfunction in high producing dairy cows in Belgium: A field study. Theriogenology 2000;53:841-857. 21. Pursley JR, Wiltbank MC, Stevenson JS, Ottobre JS, Garverick HA, Anderson LL. Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronizated ovulation or synchronized estrus. J Dairy Sci 1997;80:295-300. 22. Savio JD, Boland MP, Hynes N, Roche JF. Resumption of follicular activity in the early post-partum period of dairy cows. J Reprod Fert 1990;88:569-579. 23. Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle of heifers. J Reprod Fertil 1988;83:663-671. 24. Savio JD, Thatcher WW, Badinga L, de la Sota RL, Wolfenson D. Regulation of dominant follicle turnover during the oestrous cycle in cows. J Reprod Fertil 1993;97: 197-203. 25. Savio JD, Thatcher WW, Morris GR, Entwistle K, Drost M, Mattiacci MR. Effects of induction of low progesterone concentrations with a PRID on follicular turnover and fertility in cattle. J Reprod Fertil 1993;98:77-84. 26. Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol Reprod 1988;39:308-317. 27. Sirois J, Fortune JE. Lengthening the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endocrinology 1990;127:916-925. 28, Stock AE, Fortune JE. Ovarian follicular dominance in cattle: relationship between prolonged growth of the ovulation follicle and endocrine parameters. Endocrinology 1993;132:1108-1114. 29. Taylor C, Rajamahendran R, Walton JS. Ovarian follicular dynamics and plasma luteinizing hormone concentrations in norgestomet-treated heifers. Anim Reprod Sci 1993;32:173-184 30. Thatcher WW, Drost M, Savio JD, Macmillan KL, Entwistle KW, Schmitt EJ, De la Sota, RL, Morris GR. New clinical uses of GnRH and its analogues in cattle. Anim Reprod Sci 1993;33:27-49. 3 1. Twagiramungu H, Guilbault LA, Dufour JJ. Synchronization of ovarian follicular waves with a gonadotropin-releasing hormone agonist to increase the precision of estrus in cattle: areview. JAnimSci 1995;73:3141-3151.
PERSISTENT OVARIAN FOLLICLES IN DAIRY COWS: A THERAPEUTIC APPROACH F. Lopez-Gatius, la P. Santolaria,2 J. Yaniz, ’ J. Ruthant
and M. Lopez-Bejar
‘Animal Production, University of Lleida; 2Animal Production, University of Zaragoza 3Veterinarian Anatomy and Embryology, Autonomous University of Barcelona, Spain Received for publication: Septenker 21 , 2000 Accepted: April 9, 2001 ABSTRACT Anestrus is common during the postpartum period in high-producing dairy cows. In a previous investigation, we were able to diagnose persistent follicles of 8 to 12 mm in anestrous cows. This report describes 2 consecutive studies. The objectives of the first were to 1) assess the association of persistent follicles with anestrus; and 2) evaluate 2 therapeutic treatments. In the second study, we compared the effectiveness of the best treatment established in Study 1 with the Ovsynch protocol. For Study 1, anestrous cowswere considered to have a persistent follicle if it was possible to observe a single follicular structure > 8 mm in the absence of a corpus luteum or a cyst in 2 ultrasonographic examinations performed at an interval of 7 d. At diagnosis (Day 0), cows were assigned to 1 of 3 treatment groups. Cows in Group GnRH/PGF (n= 17) were treated with 100 ug GnRH im, and 25 mg PGFh im on Day 14. Cows in Group PRID (n=lS) were fitted with a progesterone releasing intravaginal device (PRID, containing 1.55 g of progesterone) for 9 d and were given 100 ng GnRH im at the time of PRID insertion, and 25 mg PGFh im on Day 7. Cows in Group Control (n=lS) received no treatment. The animals were inseminated at observed esnus and were monitored weekly by ultrasonogmphy until AI or 5 weeks from diagnosis. Blood samples were also collected on a weekly basis for progesterone determination. The mean size of persistent follicles on Day 0 was 9.4 + 0.04 mm. Progesterone levels were < 0.2 ng/mL during the first 35 d in 16 of 18 Control cows. Cows in the PRID group showed a lower persistent follicle rate (16.7% < 70.6% < 88.9%; P < 0.0001; PRID vs GnRH/PGF vs Control, respectively); a higher estms detection rate (83.3% > 29.4% > 11.l%; P < 0.0001) and a higher pregnancy rate (27.8% > 59% > 0%; P = 0.02). For the second study, 145 cows with persistent follicles were randomly assigned to 1 of 2 treatment groups: cows in Group Ovsynch (n=73) were treated with 100 lrg GnRH im on Day 0,25 mg PGFh im on Day 7, and 100 pm GnRH im 32 h later. Cows in this group were inseminated 16 to 20 h after the second GnRH dose (Ovsynch protocol). Cows in Group PRID (n=72) were treated as those in the PRID group of Study 1, and were inseminated 56 h after PRID removal. Cows in the PRID group showed a higher ovulation rate (84.8% > 8.2%; P < 0.0001); a higher pregnancy rate (34.2% > 4.1%; P < 0.0001) and lower follicular persistence rate (22.2% < 63%; P < 0.0001) than those in Ovsynch. Our results indicate that persistent follicles affect cyclic ovarian function in lactating dairy cows. Cows with persistent follicles can be successfully synchronized and time inseminated using progesterone, GnRH and PGF2a but show a limited response to treatment with GnRH plus PGF2a. 0 2001 by Elsevier Sc~ence Inc. Key words: dairy cows, ovary, persistent follicles, progesterone, G&II, PGFk Acknowledgments The authors thank Ana Burton for assistance with the English translation. aCorrespondence and reprint requests: Escuela T&c&a Superior de Ingenieria Agraria Universidad de Lleida, Avda. Alcalde Rovira Roure 177,25 198 Lleida, Spain, e-mail: [email protected] Theriogenology 56:64M59. 2001 0 2001 Elsevier Science Inc.
0093-691X/01/$-see front matter PII: SOO93-691X(01)00596-9
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INTRODUCTION There are 2 or 3 waves of follicular growth in the normal estrus cycle of cattle. Each wave involves the development of a large dominant follicle and smaller subordinate follicles. When the largest follicle reaches a mean diameter of 8.5 mm, follicular deviation begins (9). The deviation mechanism consists of the reduction or cessation of growth of the remaining follicles (subordinate follicles) while the largest follicle becomes dominant and increases in size. Luteinizing hormone appears to regulate the function of the dominant follicle (23, 26, 28). Final maturation and ovulation of the dominant follicle is associated with a rapid LH pulse frequency and follicular atresia is associated with a slow frequency (28). However, low progesterone concentrations or treatment with synthetic progestogens used to synchronize estrus in beef cattle have been associated with intermediate LH pulse frequencies (1 pulse per 1 to 2 h), maintaining estradiol production by a dominant follicle which becomes persistent (24, 28, 29). Persistence of the dominant follicles reduces fertility (2, 25, 28), and acute progesterone administration regresses induced persistent follicles, improving subsequent conception rates (1, 17). Follicles dominant for an extended period of time were called “prolonged“ or “persistent” dominant follicles (27), and the term “persistent follicles” is used extensively in beef cattle (13). Induced persistent follicles are smaller than typical ovarian cysts (5), and may be considered a cause of anestrus (19). Anestrus, a common state during the postpartum period in high-producing dairy cows, has major economic implications since it increases the length of the calving interval. Anestrus is either a failure to exhibit estrus behavior in cyclic cows - the most frequent finding in cows with luteinized cysts - or is due to inactive ovaries. Lactating dairy cows are diagnosed as having inactive ovaries when they show no behavioral signs of estrus, accompanied by failure to detect a corpus luteum or cyst at 2 consecutive examinations per rectum performed at an interval of 7 d (16). In a previous study (15), 13% of cows were diagnosed as having inactive ovaries (range 12 to 14.2% in different farms). These animals were then subjected to transrectal ultrasonography on two more occasions (7 d apart) after the rectal examinations. One or 2 follicles of 8 to 12 mm were detected in all animals during the first ultrasonography. Similar structures persisted in each ovary 7 d later and were considered persistent ovarian follicles and thus a possible cause of anestrus. As far as we are aware, this is the only report of clinical cases of spontaneous persistent ovarian follicles in lactating dairy cows, and no therapeutic approaches for this condition have been described in the literature. On these grounds, we undertook two consecutive studies. The objectives of the first study were to 1) assess the association of persistent follicles with anestrus; and 2) evaluate the 2 treatments: GnRH plus PGF2cr administered 14 d later; and progesterone for 9 d, with GnRH given at the start of treatment and PGF2a given on Day 7. The second study was designed to compare reproductive performance after timed AI of cows with persistent follicles that were treated with GnRH, progesterone, and PGF2a as in Study 1 with that of animals treated with GnRH, PGF2a on Day 7 and GnRH 32 h later (Ovsynch protocol). MATERIALS AND METHODS Study 1 All drugs were purchased from Sanoti Salud Animal (Barcelona, Spain) unless otherwise indicated.
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Animals. This study was performed from September 1998 to May 1999 on a commercial dairy herd in northeastern Spain. Mean annual milk production of the herd for this period was 9840 kg per cow. The cows were milked 3 times daily and were kept in open stalls. The animals were grouped according to production and age such that one group was formed by cows in their first lactation period. The study was performed on cows from this group. Cows with clinical disorders detected on postpartum examination or during the study were removed from the program. The disorders included mastitis, lameness, digestive disorders, abnormal genital discharges and pathological abnormalities of the reproductive tract detectable on palpation per rectum. Thus, the study population was formed by 53 cows in their first lactation period. The herd was maintained on a weekly reproductive health program. The reproductive tract of each animal was examined by palpation per rectum within 43 to 49 d postpartum to check for normal uterine involution and ovarian structures. For ultrasonographic examination, we selected cows with a single follicular structure of 8 to 15 mm, no corpus luteum or cyst, and no estrus signs in 7 d. These animals were then subjected to 2 ultrasonographic examinations at an interval of 7 d. A cow was considered to have a persistent follicle when the follicular structure detected by rectal palpation could be observed in both ultrasonographic examinations in the absence of a corpus luteum or cyst. Ultrasonography. Cows were diagnosed as having a persistent follicle 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 follicles was measured using the in-built electronic caliper after freezing the image on screen. The largest and the smallest diameters were measured and the mean diameter was then recorded. Cows were submitted to ultrasonographic examinations at 7 d intervals until AI or 5 weeks of diagnosis. The ovaries of inseminated cows were also examined ultrasonographically 10 d after Al. All examinations were performed by the same operator. Treatments and artilicial insemination. At the time of diagnosis of persistent follicles (Day 0), the cows were alternately assigned to 1 of 3 treatment groups (Figure 1) according to the chronological order of their calving data. Cows in Group GnRH/PGF (n= 17) were treated with GnRH (100 pg, im; Cystorelyn) on Day 0, and given one luteolytic dose of PGFla (25 mg im; Enzaprost) intravaginal 9 d without the time of received no
on Day 14. Cows in Group PRID (n=18) received a progesterone releasing device (PRID, containing 1.55 g of progesterone) on Day 0 that was maintained for the estradiol benzoate capsule. These animals were also given 100 ug GnRH im at PRID insertion and 25 mg PGF za im on Day 7. Cows in Group Control (n=l8) treatment.
All animals were inseminated semen from a single ejaculate.
at observed estrus by the same practitioner
using frozen
Progesterone analysis. Blood samples were taken just before the second (Day 0) and at all the following ultrasonographic examinations. A blood sample was also obtained IO d after AI in inseminated cows, All blood was collected into heparinized vacuum tubes from the coccygeal vein. Plasma was separated by centrifugation within 2 h and then was stored at -20°C until assayed. Progesterone was measured using solid-phase RIA kits containing antibody-coated
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tubes, ‘251-labeled progesterone and rabbit antiserum (CS Bio International, Gif-Yvette, France). The RIA method was previously validated as described for the cow by Guilbault et al. (11). 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 coefficient of variation was 9.6%. Plasma progesterone concentrations were used to classify cows as showing (2 lng/mL) or not showing (< lng/mL) luteal activity.
Group GnRHlPGF Day 14 Day 0 ()______ _____________________________~_______-___o GnRH PGF2,
AI at estrus
Group PRID Day 0 0 PRID insertion + GnRH
Day 7 1 PGP2,
Day 9 ______________________ 0 PRlD removal AI at es&us
Group Control Day 0 0 _____________________--_________________________
AI at%trus
Ultrasonographic examinations and blood sampling times Day 7... At 7 d intervals until AI... Day 0 ~_____________-____-+--_____________________-________ Figure 1. Schematic summary of treatment groups, ultrasonographic sampling times.
10 d after AI
examinations
and blood
Detection of estrus and pregnancy diagnosis. The animals were inspected for signs of estrus (standing to be mounted) at least 4 times throughout the day. Cows were inseminated approximately 8 to 10 h after the first signs of estrus were observed. Pregnancy diagnosis was performed by palpation per rectum at 34 to 40 d postinsemination. Data analysis. The follicular persistence rate was defined as the percentage of cows maintaining an 8 to 15 mm follicle on Day 35. The estrus detection rate was taken as the percentage of cows showing estrus between Days 0 and 35. The pregnancy rate was defined as the number of cows that became pregnant expressed as a percentage of the total number of cows in each group at the same period.
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Treatment regimes were compared in terms of follicular persistence, estrus detection and pregnancy rates using the Chi-square test. Data from Control cows were used to determine the possible association of follicular persistence with anestrus. Study 2 Animals, treatments and timed insemination. The second study was performed in another dairy farm, less than 30 km away from the first, over the period May to September 1999. Milking and management practices were similar to those of the previous farm. Mean annual milk production was slightly over 10,000 kg per cow. The cows were subjected to the same reproductive program and follicular persistence was diagnosed as described for Study 1. The study population was formed by 145 cows with persistent follicles that were free of other clinical disorders. This time, the lactation number was 1.8 and ranged from 1 to 3 lactations. Cows were paired by lactation number and 1 cow from each pair was randomly assigned to receive one of two treatments: the Ovsynch program designed by Pursley et al. (21); and progesterone, GnRH and PGF2a as described in Study 1. Cows in Group Ovsynch (n=73) were treated with GnRH (100 pg im; Cystorelyn) on Day 0 and were given one luteolytic dose of PGF2, (25 mg im; Enzaprost) on Day 7, and 100 pm GnRH im 32 h later. The cows were then inseminated 16 to 20 h after the second GnRH injection. Cows in Group PRID (n=72) were treated as those in the PRID group of Study 1 and were inseminated 56 h after PRID removal. All animals were inseminated by the same practitioner with frozen semen from a single ejaculate without visual detection of estrus. Two cows lost their PRID device and were excluded from the study. Collection of data after insemination. The ovaries of inseminated cows were examined ultrasonographically 10 d after AI. 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 34 to 40 d postinsemination. Open cows with a corpus luteum were included in a synchronization program. Open cows with a single follicle larger than 8 mm with no corpus luteum or cyst were subjected to a further ultrasonographic examination the next week to determine the presence of a persistent follicle. Data analysis. The effect of treatment was evaluated in terms of the dependent variables defined in Table 1. The independent variables were the treatment groups.
Table 1. Definition of dependent variables. Variable Definition Number of cows with at least 1 corpus luteum on Day 11 after AI as Ovulation rate a percentage of the total number of cows in each group Number of pregnant cows after first AI as a percentage of the total Pregnancy rate number of cows in each group Cows returning to estrus Number of cows returning to estrus 8 to 30 d postinsemination as a percentage of the total number of cows in each group Follicular persistence Number of cows with a persistent follicle 7 d after pregnancy diagnosis as a percentage of the total number of cows in each group
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Treatment regimes were compared using the Chi-square test. Values corresponding to both studies are expressed as the mean f standard deviation (SD). RESULTS Study 1 The mean number of days from calving to diagnosis of persistent follicles (Day 0) was 61 .S f 2.1 d, ranging from 57 to 63 d. Cows showed no luteal activity on Day 0. Mean plasma progesterone concentration was 0.07 * 0.03 ng/mL, ranging from 0.05 to 0.2 ng/mL. Plasma progesterone concentrations were I 0.2 ng/mL throughout the study for Control cows with persistent follicles. Mean persistent follicle sizes at Day 0 were 9.6 rt 0.05, 9.4 f. 0.06 and 9.3 + 0.06 mm for cows in the GnRH/PGF, PRID and Control groups respectively. All cows showing no signs of estrus had persistent follicles that were of similar size throughout the study. One cow in the GnRH/PGF group showed estrus signs during the first week after GnRH treatment. The remaining 16 cows showed no luteal activity on Day 7. Regression of follicles and luteal activity were registered in 5 cows on Day 14. Follicular regression was defined as the absence of follicles larger than 8 mm. Four of these cows reached estrus during the following week. The cow showing luteal activity which subsequently failed to display signs of estrus developed a new persistent follicle 7 d after prostaglandin treatment. In the remaining 11 cows, follicles persisted throughout the 5 week study period with the exception of one cow in which a new persistent follicle developed in the contralateral ovary, the first having regressed in the third week. Only I cow became pregnant after prostaglandin treatment. The average plasma progesterone concentration in the PRID group was 2.8 f 0.3 ng/mL progesterone, ranging from 1.5 to 4.2 ng/mL, on Day 7 after PRID insertion. Persistent follicles regressed in 15 of the 18 cows during the first week. These 15 cows (83%) showed estrus signs 2 to 4 d after PRID withdrawal: 13115 (72%) cows reached estrus at 48 h, 1 at 60 h and the remaining cow at 96 h. The remaining 3 cows maintained their persistent follicles during the 5 weeks of study. Five cows became pregnant. Only 2 Control group cows showed estrus after diagnosis, on Days 20 and 26 respectively. Persistent follicles were still present in the 16 remaining anestrous cows throughout the 5-week study period. However, one cow developed an additional persistent follicle in the same ovary during the fourth week and both follicles were present on Day 35. A further cow developed a persistent follicle in the contralateral ovary after regression of the first follicle in the fourth week. No cows became pregnant. Table 2 shows follicular persistence, estrus detection and pregnancy rates for cows in the 3 treatment groups. A significant effect was shown by treatment on all the variables.
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Table 2.
Group
655
Effect of treatment with GnRH/PGF or PRID on cows 35 days after they had been diagnosed with persistent follicles. Results are measured by follicular persistence, estrus and pregnancy rates.
Estrus detection rate Follicular persistence Pregnancy rate (%)’ (%)h rate (%)a GnRH/PGF 17 29.4 70.6 5.9 PRID 18 16.7 83.3 27.8 Control 18 88.9 11.1 0 GnRH/PGF = 100 ug GnRH im on Day 0, and 25 mg PGFln im on Dayl4. PRID = 1.55 g intravaginal progesterone for 9 d, plus 100 ug GnRH im on Day 0 followed by 25 mg PGFla im on Day 7. Control = untreated cows. a.hProportions were different (P < 0.0001) when compared in a 3x2 contingency table using the Chi-square test. ‘Proportions were different (p = 0.02) when compared in a 3x2 contingency table using the Chi-square test. n
Ovulation was confirmed by the presence of a corpus luteum inseminated cows 10 d after AI. No double ovulations were recorded.
and luteal activity
in all
Study 2 The mean number of days recorded from calving to diagnosis was 60.2 ?_ 1.6 d, ranging from 57 to 63 d.
of persistent
follicles
(Day 0)
A significant effect was shown by treatment on the ovulation rate, pregnancy rate and the percentage of open cows with persistent follicles 7 d after pregnancy diagnosis (Table 3). The proportion of cows returning to estrus was unaltered by treatment.
Table 3. Effect of treatment regimen on ovulation rate, pregnancy rate, percentage of cows returning to estrus and percentage of open cows with persistent follicles 7 d after pregnancy diagnosis. Effects Ovsynch (n=73) PRID (n=72) P Ovulation rate (%) 8.2 84.8
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Three open cows in the Ovsynch group and 2 in the PRID group were found to have cystic ovaries after pregnancy diagnosis. Six open cows in Ovsynch and 1 in PRID had 2 persistent follicles of similar size on Day 7 d after pregnancy diagnosis. Follicular persistence was also recorded in the last 7 cows. DISCUSSION The effects of spontaneous persistent follicles on cyclic ovarian function in lactating dairy cows is documented for the first time in Study 1. Since progesterone levels were stable and very low for 35 d in 16 out of 18 Control cows, we believed that anestrus was most probably associated with persistent follicles. In a previous study including data from 3 dairy farms (15), all anestrous cows diagnosed as having inactive ovaries had persistent follicles with no corpora lutea or cysts. Indeed, an extensive epidemiological review of the incidence of persistent follicles in anestrous cows would be of major interest. The development of persistent follicles in lactating dairy cows may result from a failure to resume ovarian activity in the postpartum period. Selection of the first dominant follicle can occur as early as 7 to 15 d after calving, and 1 to 3 follicular waves can be observed before first ovulation, which usually occurs between 15 and 2.5 d post partum (22). Hereafter the cows resume their cyclic ovarian function. Nevertheless, the length of the postpartum anovulatory period can be affected by a number of factors such as a negative energy balance (4), milk production, age, calving season and reproductive diseases (10, 20). The development of anovulatory or cystic follicles may in these cases prolong the interval until first ovulation to 40 or 50 d postpartum (3). In the present study, although there was no interest in determining the time of first ovulation, our data indicate that cows with persistent follicles can exceed 90 d postpartum before they return to cyclicity. The mean number of days from calving to diagnosis of persistent follicles was 61.8 + 2.1 d, and these persisted for 35 d in 16 of 18 Control cows. It is possible that cows with persistent follicles were recovering from extended periods of negative energy balance after calving. Moreover, the metabolic demands of lactation could also hinder final follicular maturation and ovulation. In Study 1, follicular persistence was characterized by the presence of a follicular structure similar in size to that of a dominant follicle (> 8 mm). This follicle was morphologically dominant for an extended period of time. The main clinical effect of the persistent follicles, anestrus, may be compared with that of luteinized cysts. Three phenomena described for ovarian follicular cysts (7, 12) were also observed in Control cows with persistent follicles: persistence (15 cows), spontaneous recovery (2 cows spontaneously ovulated) and turnover (1 cow developed a new persistent follicle in the contralateral ovary after regression of the first). Replacement by a new follicle in the contralateral ovary was also observed in one cow from the GnRH/PGF group. Due to our work conditions, in which only a single ultrasonographic examination was performed per week, it is possible that more follicles were replaced by new persistent follicles in the same ovary, however, persistence for an extended period appears to be the foremost feature of persistent follicles. In a recent study (19), in which ultrasonography was conducted each day for 26 d and then on alternate days, induced persistent follicles remained detectable up to 52 d. The dynamic nature and turnover of spontaneous persistent ovarian follicles in dairy cows requires further investigation.
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The administration of GnRH and its synthetic analogues acutely increases LH and FSH levels in the peripheral circulation for a 2 to 4 h period (6). In cyclic cows, GnRH induces ovulation or atresia of the dominant follicle, depending on the stage of follicular development and the emergence of a new follicular wave within 3 to 4 d after treatment (30, 3 1). Exogenous GnRH has also been extensively used for ovarian cyst treatment (8). Cysts respond to GnRH by luteinization of the cystic structure and the success rate of treatment is currently around 80% (8, 30). However, in the present study, the administration of GnRH induced ovulation or atresia in only 6 of 17 cows (35%) and no cows showed luteal activity on Day 7 in the GnRH/PGF group (Study 1). Only 6 of 73 cows (8%) ovulated after the second GnRH injection in the Ovsynch group (Study 2). Furthermore, follicular persistence was recorded in 46 of 73 cows (63%) 40 d after Ovsynch treatment (Study 2). These results indicate a poor response of cows with persistent follicles to GnRH. It would appear that factors other than LH surge may induce extended periods of anovulatory activity in the presence of persistent follicles. Ovarian cysts are anovulatory follicle-like structures greater than 2.5 cm that persist for 10 d or more in the absence of a corpus luteum (8). Although smaller in size, persistent follicles would be expected to respond to treatment in the same way as follicular cysts. Thus, two types of treatment approaches adapted from follicular cyst therapy were tested in Study 1. Treatment of follicular cysts with GnRH and with a luteolytic agent 14 d later (GnRH/PGF group), has been shown to be an efficient method for cyst recovery (8, 18). The treatment regime progesterone for 9 d, GnRH on Day 0, and PGF2a on Day 7 (PRID group) has also been proposed as appropriate treatment for cysts (30). Here, best results were associated with PRID treatment, which gave rise to the highest overall estrus detection rates (83%), a high degree of synchrony (72% of cows started estrus after 48 h of PRID removal), and a lower follicular persistence rate and higher pregnancy rate than GnRH/PGF treatment. This treatment protocol permitted timed insemination at 56 h in Study 2. The administration of progesterone and GnRH in cows in the PRID group (Study 1) induced the regression of persistent follicles in most cows (15 of 18) during the first week. These findings agree in part with those reported for beef cattle. In beef cows that developed persistent ovarian follicles after estrus synchronization with progestogens, the acute administration of progesterone decreased LH secretion (17) atresia of persistent follicles and recruitment of a new dominant follicle (1, 17, 28). Progesterone was probably the most efficient component of the PRID protocol used in the present study. In Study 2, we compared the reproductive performance of lactating dairy cows following the Ovsynch protocol (Ovsynch group) with the administration of progesterone, GnRH and PGF2a (PRID group). Best results were also associated with PRID treatment, with a higher ovulation rate (85% after PRID removal) and pregnancy rate (34%) recorded along with a lower follicular persistence rate 40 d after treatment (22%). The pregnancy rate was similar to that of lactating dairy cows in natural estrus (36%) in the geographical area of the study (14). In conclusion, the present findings indicate that lactating dairy cows with persistent follicles can be successfully synchronized and time inseminated using a protocol that combines progesterone, GnRH and PGF2a. In contrast, persistent follicles were scarcely responsive to treatment with GnRH plus PGF2o.
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REFERENCES 1. Anderson LH, Day ML. Acute progesterone administration regresses persistent dominant follicles and improves fertility of cattle in which estrus was synchronized with melengestrol acetate. J Anim Sci 1994;72:2955-2961. 2. Austin EJ, Mihm M, Ryan MP, Williams DH, Roche JF. Effect of duration of dominance of the ovulatory follicle on onset of estrus and fertility in heifers. J Anim Sci 1999;77:22 192226. 3. Beam SW, Butler WR. Energy and ovarian follicle development prior to the first ovulation postpartum in dairy cows receiving three levels of dietary fat. Biol Reprod 1997;56:133142. 4. Beam SW, Butler WR. Effects of energy balance on follicular development and first ovulation in postpartum dairy cows. J Reprod Fertil 1999;Suppl 54:41 l-424. 5. Bigelow KL, Fortune JE. Characteristics of prolonged dominant versus control follicles: follicle cell numbers, steroidogenic capabilities, and messenger ribonucleic acid for steroidogenic enzymes. Biol Reprod 1998;58:1241-1249. 6. Chenault JR, Kratzer DD, Rzepkowski RA, Goodwin MC. LH and FSH response of holstein heifers to fertirelin acetate, gonadorelin and buserelin. Theriogenology 1990;34:81-98. 7. Cook DL, Smith CA, Parfect JR, Youngquist RS, Brown EM, Garverick HA. Fate and turnover rate of ovarian follicular cysts in dairy cattle. J Reprod Fertil 1990;90:37-46. 8. Garverick HA. Ovarian follicular cysts in dairy cows. J Dairy Sci 1997;80:995-1004. 9. Ginther OJ. Selection of the dominant follicle in cattle and horses. Anim Reprod Sci 2000;60-61:61-79. 10. Grohn YT, Rajala-Schultz PJ. Epidemiology of reproductive performance in dairy cows. Anim Reprod Sci 2000;60-61:605-614. 11. Guilbault LA, Roy GL, Grass0 F, Matton P. Influence of pregnancy on the onset of oestrus and luteal function after prostaglandin-induced luteolysis in cattle. J Reprod Fertil 1988; 84:461-468. 12. Hamilton SA, Garverick HA, Keisler DH, Xu ZZ, Loos K, Youngquist RS, Salfen BE. Characterization of ovarian follicular cysts and associated endocrine profiles in dairy cows. Biol Reprod 1995;53:890-898. 13. Kinder JE, Kojima FN, Bergfeld EGM, Wehrman ME, Fike KE. Progestin and estrogen regulation of pulsatile LH release and development of persistent ovarian follicles in cattle. J Anim Sci 1996;74: 1424-1440. 14. Lopez-Gatius F. Reproductive performance of lactating dairy cows treated with cloprostenol, hCG and estradiol benzoate for synchronization of es&us followed by timed AI. Theriogenology 2000;54:551-558. 15. Lopez-Gatius F, Lopez-B&jar M, Rutllant J, Yaniz J, Santolaria P. Persistent follicles in high-producing dairy cows Proc EETA Annual Meeting, Lyon France, 1999; 190 abst. 16. Markusfeld 0. Inactive ovaries in high-yielding dairy cows before service: Aetiology and effect on conception, Vet Ret 1987; 12 1: 149-I 53. 17. McDowell CM, Anderson LH, Kinder JE and Day ML. Duration of treatment with progesterone and regression of persistent ovarian follicles in cattle. J Anim Sci 1998;76: 850-855. 18. Nanda AS, Ward WR, Williams PCW, Dobson H. Retrospective analysis of the efficacy of different hormone treatments of cystic ovarian disease in cattle. Vet Ret 1988;122: 155-l 58.
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19. Noble KM, Tebble JE, Harvey D, Dobson H. Ultrasonography and hormone profiles of persistent ovarian follicles (cysts) induced with low doses of progesterone in cattle. J Reprod Fertil 2000;120:361-366. 20. Opsomer G, Griihn YT, Hertl J, Coryn M, Deluyker H, de Kruif A. Risk factors for post pat-turn ovarian dysfunction in high producing dairy cows in Belgium: A field study. Theriogenology 2000;53:841-857. 21. Pursley JR, Wiltbank MC, Stevenson JS, Ottobre JS, Garverick HA, Anderson LL. Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronizated ovulation or synchronized estrus. J Dairy Sci 1997;80:295-300. 22. Savio JD, Boland MP, Hynes N, Roche JF. Resumption of follicular activity in the early post-partum period of dairy cows. J Reprod Fert 1990;88:569-579. 23. Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle of heifers. J Reprod Fertil 1988;83:663-671. 24. Savio JD, Thatcher WW, Badinga L, de la Sota RL, Wolfenson D. Regulation of dominant follicle turnover during the oestrous cycle in cows. J Reprod Fertil 1993;97: 197-203. 25. Savio JD, Thatcher WW, Morris GR, Entwistle K, Drost M, Mattiacci MR. Effects of induction of low progesterone concentrations with a PRID on follicular turnover and fertility in cattle. J Reprod Fertil 1993;98:77-84. 26. Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol Reprod 1988;39:308-317. 27. Sirois J, Fortune JE. Lengthening the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endocrinology 1990;127:916-925. 28, Stock AE, Fortune JE. Ovarian follicular dominance in cattle: relationship between prolonged growth of the ovulation follicle and endocrine parameters. Endocrinology 1993;132:1108-1114. 29. Taylor C, Rajamahendran R, Walton JS. Ovarian follicular dynamics and plasma luteinizing hormone concentrations in norgestomet-treated heifers. Anim Reprod Sci 1993;32:173-184 30. Thatcher WW, Drost M, Savio JD, Macmillan KL, Entwistle KW, Schmitt EJ, De la Sota, RL, Morris GR. New clinical uses of GnRH and its analogues in cattle. Anim Reprod Sci 1993;33:27-49. 3 1. Twagiramungu H, Guilbault LA, Dufour JJ. Synchronization of ovarian follicular waves with a gonadotropin-releasing hormone agonist to increase the precision of estrus in cattle: areview. JAnimSci 1995;73:3141-3151.