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Effect of milk production on the incidence of double ovulation in dairy cows
ELSEVIER
EFFECT OF MILK PRODUCTION ON THE INCIDENCE DOUBLE OVULATION IN DAIRY COWS
OF
P.M. Frickc? and M.C. Wiltbank Department of Dairy Science, University of Wisconsin-Madison Madison, WI 53706 USA Received for publication: Accepted:
July
8,
October
1 ggg
5,
1999
ABSTRACT To determine the effect of parity and milk production on the incidence of double ovulation, the synchronization of ovulation, using GnRH and prostaglandin Fza followed by timed AI (Ovsynch@), was initiated at a random stage of the estrous cycle in lactating Holstein cows (n=237). Ovulatory response at 48 h after the second GnRH injection and conception rate at 28 d post AI were determined by transrectal ultrasonography. Ovulation was synchronized in 84% of cows receiving the Ovsynch@ protocol. Of the synchronized cows, 14.1% exhibited a double ovulation and 47.6% conceived. Conception rate tended to be greater (FO.08) for cows exhibiting double (64.0%) rather than single ovulation (45.2%). To determine the effect of milk production on the incidence of double ovulation, cows were classified into low (I 40 kg/d) or high (> 40 kg/d) milk production groups based on the average milk production of 40.5 f 0.8 kg/d collected 2 d before AI. Although the incidence of double ovulation tended to increase linearly (FO.09) with increasing parity, the incidence of double ovulation was nearly 3-fold greater (P
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Key words: Double ovulation, twinning, ovarian cysts, dairy cows, Ovsynch Acknowledgements We thank Blue Star Dairy, De Forest, WI, for use of their animals and facilities, The Pharmacia-Upjohn Co., Kalamazoo, MI, for providing Lutalyse, and Merial, Ltd., for providing Cystorelin. This research was supported in part by a competitive grant from the National Association of Animal Breeders to P.M.F. and M.C.W. ?Zorrespondence and reprint requests. Phone: (608) 263-4596; Fax: (608) 263-9412; Email: [email protected]. Theriogenology 52:1133-l 143, 1999 0 1999 by Elsevier Science Inc.
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INTRODUCTION Follicular growth in cattle occurs as regular periods of follicular growth, dominance and atresia, described as follicular waves (14). Normally, only 1 follicle within each wave is selected to become dominant and acquire ovulatory capacity through a process we have termed deviation (14,31). Deviation, therefore, provides an intrinsic mechanism that restricts the number of ovulatory follicles present on the ovaries at any given time during the bovine estrous cycle to a single follicle. Occasionally, however, this selection mechanism is abrogated and 2 follicles within the same follicular wave undergo deviation, resulting in a phenomenon termed codominance. Because both co-dominant follicles have undergone deviation and thereby have acquired dominance and ovulatory capacity, an endogenousor exogenous ovulatory stimulus that occurs when codominant follicles are present will result in a double ovulation. If the developmental events from fertilization to parturition occur without interruption for both oocytes from the co-dominant follicles, dizygous twins will result. Twinning is an unavoidable outcome of reproduction in dairy cattle and is undesirable in a dairy operation because it reduces overall profitability and reproductive efficiency (3,8) by increasing average days open and services per conception of the dam during the subsequent lactation (25). Cows calving twins have a higher risk for stillbirth, retained placenta, metritis, displaced abomasum, ketosis, and aciduria than cows calving singletons (2 1,25,28). Incidences of abortion, neonatal calf mortality, reduced birth weight, and retained placenta also are greater among twin versus singleton calves, probably due to reduced gestation lengths and increased incidences of dystocia among cows calving twins (6,9,25,28). Culling rates also are greater for cows calving twins (8) probably due to the aforementioned adverse effects of twinning. Because most twins in cattle are dizygous (9,16,30), development of management strategies to alter the incidence of twinning in dairy cattle requires a thorough understanding of the factors that regulate follicular selection and double ovulation. Few recent studies have reported the incidence of double ovulation in dairy cattle. Previous studies from the same laboratory, in which the number of corpora lutea (CL) present 7 to 13 d after a spontaneous estrus was determined by using palpation per rectum, reported double ovulation rates of 13.1% (18) and 5.4% (20). However, becausethere was considerable disparity in the reported incidence of double ovulation between these studies, the authors questioned their technical ability to detect multiple CL using palpation per rectum, particularly in their earlier study. Transrectal ultrasonography provides an accurate method for evaluating ovarian events in cattle (15). In addition, a novel protocol that synchronizes the time of ovulation in lactating dairy cows (Ovsynch@; 29) provides an efficient method for analyzing the incidence of double ovulation in a large number of cows. The primary objective of this study was to determine the effect of parity and milk production on the incidence of double ovulation in lactating dairy cows. A secondary objective was to determine the effect of the Ovsynch@ protocol in cows with ovarian cysts. Our hypothesis was that the incidence of double ovulation in lactating dairy cows would increase as parity and milk production increased.
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MATERIALS AND METHODS Source of Data Results from the present study involved the assessmentof data collected from a previous experiment in which lactating Holstein cows underwent timed AI after synchronization of ovulation using Ovsynch@(12). Neither the objective nor the hypothesis of the present study was considered in the previous experiment. Animals and Synchronization of Ovulation Primiparous and multiparous lactating Holstein cows (n=237) from a commercial dairy herd of 600 lactating Holstein cows located in south-central Wisconsin, were blocked by parity and days in milk and were assigned randomly to 1 of 2 treatment groups for synchronization of ovulation. For both treatment groups, synchronization of ovulation was initiated at a random stage of the estrous cycle using intramuscular injections of GnRH (Cystorelin; Merial, Ltd., Iselin, NJ) and PGF2, (Lutalyse; The Pharmacia & Upjohn Co., Kalamazoo, MI) as follows: Day 0, GnRH; Day 7, PGF2,; Day 9, GnRH. Ovulation was synchronized with 25 mg PGF2, plus either 100 pg (n = 119; Group 1) or 50 pg (n = 118; Group 2) GnRH per injection. Treatment (i.e., GnRH dose) had no effect on any experimental endpoint,including the conception rate (41 vs 41.1% for the 100 pg and 50 ug treatments, respectively; 12); the double ovulation rate (13.9 vs 14.3%; 12); or the incidence of ovarian cysts (10.9 vs 11.0% for the 100 pg and 50 pg treatments, respectively). Therefore, data from cows in both treatment groups were combined to evaluate the effect of parity and milk production on the incidence of double ovulation. Although some of the cows in this herd received exogenous bST as part of the management scheme of this farm, records of bST use were unavailable and were not included in this analysis. Cows were milked 3 times daily, and the rolling herd average of the farm exceeded 10,000 kg of milk throughout the duration of the study, which was conducted as 3 replicates beginning in July and September of 1997 and in March of 1998. Each replicate was completed 66 d after initiation. Milk production (mean + SEM) of the cows used for this study averaged 40.5 f 0.8 kg/d (range = 9.1 to 72.1 kg/d) based on milk test weights recorded 2 d before AI. All cows underwent a timed AI within 12 to 18 h after the second GnRH injection. The herd manager, who remained unaware of the treatment group to which each cow was assigned, chose service sires for each mating as part of the standard reproduction program used by the farm. For each replicate, AI was performed by 4 to 5 herd personnel who also remained unaware of the treatment group to which each cow was assigned. Mean (+ SEM) days in milk of the cows at AI was 139.3 + 4 d (range = 62 to 392 d). Synchronization Rate, Double Ovulation Rate, and Ovarian Cysts Ovarian structures were monitored by using an ultrasound machine equipped with a transrectal 7.5 MHz linear-array transducer (Aloka 500V; Corometrics Medical Systems, Inc., Wallingford, CT). During each ultrasound examination, a sketch of the location and diameter of all ovarian structures 1 8 mm in diameter was recorded. For each ovarian structure, 2 diameter measurements,taken at right angles, were recorded using the digital calipers equipped with the ultrasound machine; the diameters were calculated as the mean of these 2 measurements. Ovulatory response to the second GnRH injection (synchronization rate) was determined by the
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presence of 1 or more antral follicles at the time of the second GnRH injection and the absence of 1 (single ovulation) or 2 (double ovulation) of those follicles at an ultrasound examination conducted 48 h later. Synchronization rate was calculated as the number of cows that ovulated at least 1 follicle within 48 h of the second GnRH injection, expressed as a percentage of the total number of cows receiving the Ovsynch@protocol. The double ovulation rate was calculated as the number of cows that ovulated 2 follicles within 48 h of the second GnRH injection, expressed as a percentage of synchronized cows. Fluid-filled cystic structures 2 2.5 mm in diameter identified at the scanning session conducted at the time of the second GnRH injection were classified based on ultrasonographic morphology as ovarian cysts. No attempt was made to further classify cystic structures as follicular, luteal or benign based on ultrasonographic morphology. Pregnancy Diagnosis and Conception Rate Pregnancy status was determined by using the ultrasound machine and transducer described for determining synchronization rate. Visualization of a fluid-tilled uterine horn and the presence of a fetus were used as positive indications of pregnancy. For cows in Replicate 1 (n=l 10) and Replicate 2 (n=51), pregnancy status was determined at 28 d post AI. For cows in Replicate 3 (n=76), pregnancy status was determined at 33 d post AI. Pregnancy status data at 28 and 33 d post AI for cows from all 3 replicates were combined and are reported as pregnancy status data for 28 d post AI. Conception rate at 28 d post AI was calculated as the number of cows diagnosed pregnant, expressed as a percentageof cows that ovulated at least 1 follicle within 48 h of the second GnRH injection of the synchronization of ovulation protocol. Effect of Parity and Milk Production on the Incidence of Double Ovulation To determine the effect of parity and milk production on the incidence of double ovulation, cows were classified into low (< 40 kg/d) and high (> 40 kg/d) milk production groups basedon the average milk production of 40.5 f 0.8 kg/d collected 2 d before AI. Cows were also classified by parity based on their lactation number at the time of AI (1,2 or 3+ lactations). Statistical Analysis Categorical data, which included synchronization rate, conception rate, double ovulation rate, side of ovulation, milk production group and incidence of ovarian cysts were analyzed using the Mantel-Haenszel Chi-square test (32). The linear effect of parity on incidence of double ovulation was analyzed by using maximum likelihood estimates with the logistic procedure of SAS (32). RESULTS Synchronization Rate, Double Ovulation Rate, Location of Ovulation, and Conception Rate Ovulation was synchronized in 84% (199/237) of cows receiving the Ovsynch@protocol, and the conception rate at 28 d post AI for synchronized cows was 47.6% (91/191). Of the synchronized cows, 14.1% (28/199) exhibited a double ovulation, and no cows ovulated more than 2 follicles in responseto the second G&I-I injection.
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For cows exhibiting single ovulations, the incidence of ovulation tended to be higher (FO.08) for the right than for the left ovary (Table 1). Of the cows exhibiting a double ovulation, 53.6% occurred ipsilaterally (5128 cows exhibited both ovulations on the left ovary; lo/28 cows exhibited both ovulations on the right ovary), whereas 46.4% occurred contralaterally (Table 1). Conception rate at 28 d post AI tended to be higher (~~0.08) for cows exhibiting a double ovulation than for cows exhibiting a single ovulation (Table 1). Conception rate at 28 d post AI did not differ based on the side of ovulation for cows exhibiting a single ovulation. Effect of Parity and Milk Production on the Incidence of Double Ovulation Milk production data were not available for 3 of the cows in the study that ovulated in response to the Ovsynch@protocol. One of these cows exhibited a single ovulation while the remaining 2 cows exhibited a double ovulation. Data from these 3 cows were necessarily excluded from the analyses involving milk production data. The incidence of double ovulation tended to increase linearly (PrO.09) with increasing parity; however, the incidence of double ovulation was nearly 3-fold higher (PcO.05) for cows in the high versus low milk production group (Table 2). Although statistical differences within a parity group in the proportion of cows exhibiting double ovulation in the low and high milk production groups were not detected, the incidence of double ovulation was proportionately greater within each parity group for cows in the high milk production group (Table 2). Incidence of Ovarian Cysts The overall incidence of cows classified with ovarian cysts at the time of second GnRH injection of the Ovsynch@protocol was 11% (Table 3). The incidence of ovarian cysts did not differ between cows in the low (1 l/26) and high (15/26) milk production group. There were 73.1% of cows classified as having an ovarian cyst that responded to the Ovsynch@protocol by ovulating a normal size (i.e., 10 to 22 mm in diameter) follicle other than the cystic structure. Synchronization and conception rates for cows classified as cystic did not differ from that of cows classified as normal (Table 3). In no instance did a cystic structure ovulate, and only 1 of the cows classified as cystic exhibited a double ovulation. Calving Data Calving data was collected from 58 of the 91 cows diagnosed pregnant at 28 d post AI. The remaining 33 cows either experienced subsequentembryonic loss (n=19) or were culled from the herd before they calved (n=14). Of the 58 cows that calved, 11 exhibited a double ovulation in responseto the Ovsynch@protocol; 3 of the cows that exhibited a double ovulation calved twins, whereas none of the cows exhibiting a single ovulation calved twins. Thus, the overall twinning rate of the 58 cows that calved was 5.2% (3/58). Overall gender ratio of calves was 27:34 (bulls:heifers).
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Table 1. Incidence, location of ovulation, and conception rate of lactating dairy cows exhibiting single or double ovulation after a synchronized ovulation Ovulatory response
Location of ovulation(s)
Incidence (%)
Conception rate (%)
Single ovulation (number / total)
85.9 (171/199)
Left ovary (%) 43.3” (74/171)
Right ovary (%) 56.7b (97/171)
45.2” (75/l 66)
Double ovulation (number / total)
14.1 (28/199)
Ipsilateral (%) 53.6 (15/28)
Contralateral (%) 46.4 (13/28)
64.0d (16/25)
“sbIncidenceof ovulation tended to be higher (eO.08) for the right ovary. C*dConceptionrate tended to be higher (PcO.08) for cows exhibiting double ovulation. Table 2. Effect of parity and milk production (mean f SEM) on the incidence of double ovulation after a synchronized ovulation Item Milk Production (kg/day)
Milk Production (kg/d) High (> 40) Low (< 40) 50.7 + 0.7
40.5 I!I 0.8
Parity 2 (%) (number / total)
22.2 (219) 14.3 (7/49)
10.gd (8/74)
Parity 3+ (%) (number / total)
27.8 (10/36)
20.3” (12/59)
Overall (%) (number / total)
20.2b (19/94)
13.3 (26/l 96)
Parity 1 (%) (number / total)
31.1 + 0.7
Overall
a,bProportionsdiffer by x2 analysis (KO.05). Csd,eLinear increase (FO.09) in incidence of double ovulation with increasing parity.
Theriogenology Table 3. Synchronization and conception rates after synchronization of ovulation and timed AI of lactating dairy cows classified on the basis of ovarian morphology as cystic or normal Item
Ovarian morphology” Cysticb Normal
Overall
Incidence (%)
11.0 (26/237)
89.0 (2 1l/237)
Synchronization rateC(%)
73.1 (19126)
85.3 (1801211)
84.0 (1991237)
Conception rate (%) of synchronized cows
36.8 (7/19)
48.8 (84/172)
47.6 (91/191)
“No statistical difference was detected for synchronization or conception rate using x2 analysis. bDefined morphologically by using ultrasound as a fluid-filled cystic ovarian structure 2 2.5mm in diameter present at the time of the second GnEW injection of the Ovsynch’ protocol. ‘Ovulation of a follicle by 48 hours after the second GnRH injection of the Ovsynch@protocol. DISCUSSION To our knowledge, this is the first report describing a direct relationship between milk production and the incidence of double ovulation in lactating dairy cows. Based on an epidemiological analysis of data, Kinsel et al. (19) reported that peak milk production was a major risk factor for increased twinning rates in lactating dairy cows. Because most twins in dairy cattle are dizygous (9,16,30), our results are consistent with the idea that high milk production near the time of ovulation can increase the incidence of double ovulation and that this increase may subsequently result in increased twinning. The practical implication of this relationship is important, because current dairy management strategies aim to maximize milk production per cow, thereby increasing the frequency of double ovulation in the dairy cattle population as a whole. In support of this notion, the overall twinning rates reported for dairy cows in recent studies are higher than those reported previously (6). If twinning is related to milk production, this increase should not be unexpected considering the annual increases in milk production per cow that have occurred over this time. Overall, the incidence of double ovulation after a synchronized ovulation in the present study was 14.1%. Although few recent studies have reported the incidence of double ovulation, previous reports in which palpation per rectum was used to determine the number of CL present 7 to 13 d after a spontaneousestrus reported double ovulation rates of 13.1% (18) and 5.4% (20). However, becausethere was considerable disparity in the incidence of double ovulation between these studies, the technical ability to detect multiple CL using palpation per rectum, particularly in the earlier study, needs to be questioned. In our present study, we used transrectal ultrasonography to determine synchronization and double ovulation rates, a technique with a high degree of accuracy for resolving ovarian structures and events such as ovulation in cattle (15). Although we do not know whether the Ovsynch@protocol directly affected the incidence of
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double ovulation in the present study, the incidence of multiple CL before initiation of ang hormonal treatment was similar to the incidence of double ovulation in response to Ovsynch (15 vs 13%, respectively; n=156; our unpublished data). In addition, the observation that the double ovulation rate is related to milk production also argues that this methodology provides relevant information on factors affecting double ovulation. Twinning rates in dairy cattle are dramatically affected by parity, with an incidence of less than 1% for virgin heifers but approaching nearly 10% for older lactating cows (4,21,30). Although the effect of parity on twinning is riot clearly understood, increased uterine capacity of cows calving twins has been implicated (30). The incidence of double ovulation, as with the incidence of twinning, has also been reported to increase with parity in lactating dairy cows, increasing from 2.9% for Parity 1 to 5.9% for Parity 3. (20). Although our results demonstratea linear increase in the incidence of double ovulation with increasing parity, the apparent reason for this increase was that the proportion of cows with high milk production within a parity group was greater for the older cows. Thus, the physiology of high milk production in and of itself appearsto increase the incidence of double ovulation independent of either age or parity. Specific physiologic mechanisms that may predispose higher producing dairy cows to exhibit more double ovulations are not known. Increased amounts of dietary bypass protein can increase the ovulation rate and the incidence of twinning in ewes (26). High levels of bypass protein fed to lactating dairy cows may increase the incidence of double ovulation and hence the rate of twinning similar to that of the practice of “flushing” in ewes (7). Unilateral ovariectomy increased the incidence of double ovulation on the remaining ovary in dry dairy cows and pubertal dairy heifers without affecting the number of follicular waves per cycle (23). Despite this observation, FSH, LH and progesterone concentrations before and after unilateral ovariectomy did not differ, suggesting that the magnitude of endocrine changes required for 2 follicles to undergo deviation during a follicular wave may be too small to measureor may be of a short duration (23). Our recent data (35) suggest that high feed intake in lactating dairy cows may increase hepatic metabolism of ovarian steroids, similar to that reported previously in ewes (22,27). These changes in steroid metabolism may alter the endocrine environment sufficiently to allow for deviation of 2 follicles during the selection period of a follicular wave. Future studies may provide further insight into the physiological mechanisms that mediate the effects of high milk production on double ovulation rate. Although double ovulation must precede the occurrence of dizygous twins, double ovulation does not consistently result in twinning becausecows carrying twins experience high rates of embryonic loss and abortion during gestation (6). However, there were too few calvings in the present study to reliably assesspregnancy attrition in cows exhibiting double ovulation. The analysis of double ovulation rather than twinning allowed us to directly observe a physiologic event that may be affected by milk production without the confounding problems of poor fertility (11) and the potentially higher pregnancy losses (34) that occur in high producing dairy cows. Thorough analysis of the events that occur between double ovulation and twin births will be critical for understanding the factors regulating twinning in dairy cattle. A secondary objective of the present study was to evaluate the effect of Ovsynch’ on cows with ovarian cysts. The incidence of cows classified as cystic in the present study was ll%,
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similar to the reported rates of 10 to 13% in lactating dairy cows (2,lO) but less than that of problem herds, which may experience incidences of 30 to 40% over brief periods (1). Ovarian cysts in cattle commonly are defined as anovular fluid-filled structures ~25 mm in diameter that persist on the ovaries for more than 10 d (1). Because we evaluated cysts only at the time of the second GnRH injection, it was not possible to physiologically classify the cysts or assessthe their response to the first or second injection of the Ovsynch@ protocol. The physiology and etiology of ovarian cysts is poorly understood, and, although milk production has been implicated (17), it did not affect the incidence of ovarian cysts in our present study. Current hormonal therapies for ovarian cysts vary depending on the classification of the cyst. Although follicular cysts are most commonly treated with G&I-I (5,33,36), the observation that nearly 20% of cows with follicular cysts recover spontaneously (5) supports the notion that many ovarian cysts are benign and, therefore, do not interfere with the normal progression of follicular waves. Treatment of a follicular cyst with GnRH induces luteinization rather than ovulation and results in formation of a luteal cyst (13). Once formed, regression of luteal cysts can be induced by administration of PGFra (24). Other researchers (1,13) have reported the similar observation that cows with ovarian cysts frequently ovulate a normally growing follicle in response to GnRH treatment. Data from the present study also indicate that synchronization and conception rates of cows classified as cystic did not differ statistically from that of cows classified as normal. Collectively, these data support the use of Ovsynch’ as a treatment for lactating dairy cows exhibiting ovarian cysts. We conclude that milk production is the primary factor affecting the incidence of double ovulation in lactating dairy cows. Further experiments are needed to elucidate specific mechanisms whereby the physiology of increased milk production affects the number of follicles that undergo deviation and acquire ovulatory capacity during a follicular wave. Future strategies may then be developed to avoid or reduce the negative effects of twinning on dairy operations. In addition, the Ovsynch@ protocol may be an effective method for establishing pregnancy in lactating dairy cows with ovarian cysts REFERENCES 1. Archibald LF, Thatcher WW. Ovarian follicular dynamics and management of ovarian cysts. In: Van Horn HH, Wilcox CJ (eds), Large Dairy Herd Management. Champaign IL: Am Dairy Sci Assoc, 1992;199-208. 2. Bartlett PC, Ngategize PK, Kaneene JB, Kirk JH, Anderson SM, Mather EC. Cystic follicular disease in Michigan Holstein-Friesian cattle: incidence, descriptive epidemiology, and economic impact. Prev Vet Med 1986;4:15-33. 3. Beerepoot GMM, Dykhuizen AA, Mielen M, Schukken YH. The economics of naturally occurring twinning in dairy cattle. J Dairy Sci 1992;75:1044-1051. 4. Berry SL, Ahmadi A, Thurmond MC. Periparturient disease on large, dry lot dairies: interrelationships of lactation, dystocia, calf number, calf mortality, and calf sex. J Dairy Sci 1994;77(Suppl 1):379 abstr. 5. Bierschwal CJ, Garverick HA, Martin CE, Youngquist RS, Cantley TC, Brown MD. Clinical responseof dairy cows with ovarian cysts to GnRH. J Anim Sci 1975;41:1660-1665.
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6. Day JD, Weaver LD, Franti CE. Twin pregnancy diagnosis in Holstein cows: Discriminatory powers and accuracy of diagnosis by transrectal palpation and outcome of twin pregnancies. Can Vet J 1995;36:93-97. 7. Dunn TG, Moss GE. Effects of nutrient deficiencies and excesseson reproductive efficiency of livestock. J Anim Sci 1992;70:1580-1593. 8. Eddy RG, Davies 0, David C. An economic assessmentof twin births in British dairy herds. Vet Ret 1991;129:526-529. 9. Erb RE, Morrison RA. Effects of twinning on reproductive efficiency in a Holstein-Fresian herd. J Dairy Sci 1959;42:512-519. 10. Erb RE, White ME. Incidence rates of cystic follicles in Holstein cows according to 15-day and 30-day intervals. Cornell Vet 1981;71:326. 11. Faust MA, McDaniel BT, Robinson OW, Britt JH. Environmental and yield effects on reproduction in primiparous Holsteins. J Dairy Sci 1988;71:3092-3099. 12. Fricke PM, Guenther JN, Wiltbank MC. Efficacy of decreasing the dose of GnRH used in a protocol for synchronization of ovulation and timed AI in lactating dairy cows. Theriogenology 1998;50:1275-1284. 13. Garverick HA. Ovarian follicular cysts in dairy cows. J Dairy Sci 1997;80:995-1004. 14. Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Minireview. Selection of the dominant follicle in cattle. Biol Reprod 1996;55:1187-l 194. 15. Griffin PG, Ginther OJ. Researchapplications of ultrasonic imaging in reproductive biology. J Anim Sci 1992;70:953-72. 16. JohanssenI, Lindhe B, Pirchner F. Causesof variation in the frequency of monozygous and dizygous twinning in various breedsof cattle. Hereditas 1974;78:201-234. 17. Johnson AD, Legates JE, Ulberg LC. Relationship between follicular cysts and milk production in dairy cattle. J Dairy Sci 1966;49:865-868. 18. Kidder HE, Barrett GR, Casida LE. A study of ovulations in six families of HolsteinFriesians. J Dairy Sci 1952;35:436-444. 19. Kinsel ML, Marsh WE, Ruegg PL, Etherington WG. Risk factors for twinning. J Dairy Sci 1998;81:989-993. 20. Labhsetwar AP, Tyler WJ, Casida LE. Analysis of variation in some factors affecting multiple ovulations in Holstein cattle. J Dairy Sci 1963;46:840-842. 21. Marcusfeld 0. Periparturient traits in seven high dairy herds, Incidence rates, association with parity, and interrelationships among traits. J Dairy Sci 1987;70:158-166. 22. McEvoy TG, Robinson JJ, Aitken RP, Findlay PA, Palmer RM, Robertson IS. Dietaryinduced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova. Anim Reprod Sci 1995;39:89-107. 23. Mohan M, Rajamahendran R. Effects of unilateral ovariectomy on follicular development and ovulation in cattle. Theriogenology 1998;49:1059-1070. 24. Nanda AS, Ward WR, Dobson H. Retrospective analysis of the efficacy of different hormone treatments of cystic ovarian diseasein cattle. Vet Ret 1988;122:155. 25. Nielen M, Schukken YH, Scholl DT, Wilbrink HJ, Brand A. Twinning in dairy cattle: a study of risk factors and effects. Theriogenology 1989;32:845-862. 26. Nottle MB, Hynd PI, SeamarkRF, Setschell BP. Increasesin ovulation rate in lupin-fed ewes are initiated by increasesin protein digested postruminally. J Reprod Fertil 1988;84:563-566.
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27. Parr RA, Davis IF, Miles MA, Squires TJ. Feed intake affects metabolic clearance rate of progesterone in sheep.Res Vet Sci 1993;55:306-310. 28. Pfau KO, Bartlett JW, Shuart CE. A study of multiple births in a Holstein-Friesian herd. J Dairy Sci 1948;31:241-254. 29. Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGFzuand GnRH. Theriogenology 1995;44:915-923. 30. Ryan DP, Boland MP. Frequency of twin births among Holstein-Friesian cows in a warm dry climate. Theriogenology 1991;36:1- 10. 3 1. Sat-tori R, Fricke PM, Ferreira JCP, Ginther OJ, Wiltbank MC. Acquisition of ovulatory capacity by ovarian follicles during growth of follicular waves in lactating dairy cows. J Dairy Sci 1998;81(Suppl 1):223 abstr. 32. SAS Institute Inc., SAS/STAT User’s Guide, version 6. Cary NC: SAS Institute Inc, 1989. 33. Seguin BE, Convey EM, Oxender WD. Effect of gonadotropin-releasing hormone and human chorionic gonadotropin on cows with ovarian follicular cysts. Am J Vet Res 1976;37:153-157. 34. Vasconcelos JLM, Silcox RW, Lacerda JA, Pursley JR, Wiltbank MC. Pregnancy rate, pregnancy loss, and response to heat stress after AI at 2 different times from ovulation in dairy cows. Biol Reprod 1997;56(Suppl 1):140 abstr. 35. Vasconcelos JLM, Bungert KA, Tsai SJ, Wechsler FS, Wiltbank MC. Acute reduction in serum progesterone concentrations due to feed intake in pregnant lactating dairy cows. J Dairy Sci 1998;81(Suppl 1):226 abstr. 36. Whitmore HL, Hurtgen JP, Mather EC, Seguin BE. Clinical response of dairy cattle with ovarian cysts to single or repeated treatments of gonadotropin-releasing hormone. JAVMA 1979;174:1113-1115.
EFFECT OF MILK PRODUCTION ON THE INCIDENCE DOUBLE OVULATION IN DAIRY COWS
OF
P.M. Frickc? and M.C. Wiltbank Department of Dairy Science, University of Wisconsin-Madison Madison, WI 53706 USA Received for publication: Accepted:
July
8,
October
1 ggg
5,
1999
ABSTRACT To determine the effect of parity and milk production on the incidence of double ovulation, the synchronization of ovulation, using GnRH and prostaglandin Fza followed by timed AI (Ovsynch@), was initiated at a random stage of the estrous cycle in lactating Holstein cows (n=237). Ovulatory response at 48 h after the second GnRH injection and conception rate at 28 d post AI were determined by transrectal ultrasonography. Ovulation was synchronized in 84% of cows receiving the Ovsynch@ protocol. Of the synchronized cows, 14.1% exhibited a double ovulation and 47.6% conceived. Conception rate tended to be greater (FO.08) for cows exhibiting double (64.0%) rather than single ovulation (45.2%). To determine the effect of milk production on the incidence of double ovulation, cows were classified into low (I 40 kg/d) or high (> 40 kg/d) milk production groups based on the average milk production of 40.5 f 0.8 kg/d collected 2 d before AI. Although the incidence of double ovulation tended to increase linearly (FO.09) with increasing parity, the incidence of double ovulation was nearly 3-fold greater (P
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Key words: Double ovulation, twinning, ovarian cysts, dairy cows, Ovsynch Acknowledgements We thank Blue Star Dairy, De Forest, WI, for use of their animals and facilities, The Pharmacia-Upjohn Co., Kalamazoo, MI, for providing Lutalyse, and Merial, Ltd., for providing Cystorelin. This research was supported in part by a competitive grant from the National Association of Animal Breeders to P.M.F. and M.C.W. ?Zorrespondence and reprint requests. Phone: (608) 263-4596; Fax: (608) 263-9412; Email: [email protected]. Theriogenology 52:1133-l 143, 1999 0 1999 by Elsevier Science Inc.
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INTRODUCTION Follicular growth in cattle occurs as regular periods of follicular growth, dominance and atresia, described as follicular waves (14). Normally, only 1 follicle within each wave is selected to become dominant and acquire ovulatory capacity through a process we have termed deviation (14,31). Deviation, therefore, provides an intrinsic mechanism that restricts the number of ovulatory follicles present on the ovaries at any given time during the bovine estrous cycle to a single follicle. Occasionally, however, this selection mechanism is abrogated and 2 follicles within the same follicular wave undergo deviation, resulting in a phenomenon termed codominance. Because both co-dominant follicles have undergone deviation and thereby have acquired dominance and ovulatory capacity, an endogenousor exogenous ovulatory stimulus that occurs when codominant follicles are present will result in a double ovulation. If the developmental events from fertilization to parturition occur without interruption for both oocytes from the co-dominant follicles, dizygous twins will result. Twinning is an unavoidable outcome of reproduction in dairy cattle and is undesirable in a dairy operation because it reduces overall profitability and reproductive efficiency (3,8) by increasing average days open and services per conception of the dam during the subsequent lactation (25). Cows calving twins have a higher risk for stillbirth, retained placenta, metritis, displaced abomasum, ketosis, and aciduria than cows calving singletons (2 1,25,28). Incidences of abortion, neonatal calf mortality, reduced birth weight, and retained placenta also are greater among twin versus singleton calves, probably due to reduced gestation lengths and increased incidences of dystocia among cows calving twins (6,9,25,28). Culling rates also are greater for cows calving twins (8) probably due to the aforementioned adverse effects of twinning. Because most twins in cattle are dizygous (9,16,30), development of management strategies to alter the incidence of twinning in dairy cattle requires a thorough understanding of the factors that regulate follicular selection and double ovulation. Few recent studies have reported the incidence of double ovulation in dairy cattle. Previous studies from the same laboratory, in which the number of corpora lutea (CL) present 7 to 13 d after a spontaneous estrus was determined by using palpation per rectum, reported double ovulation rates of 13.1% (18) and 5.4% (20). However, becausethere was considerable disparity in the reported incidence of double ovulation between these studies, the authors questioned their technical ability to detect multiple CL using palpation per rectum, particularly in their earlier study. Transrectal ultrasonography provides an accurate method for evaluating ovarian events in cattle (15). In addition, a novel protocol that synchronizes the time of ovulation in lactating dairy cows (Ovsynch@; 29) provides an efficient method for analyzing the incidence of double ovulation in a large number of cows. The primary objective of this study was to determine the effect of parity and milk production on the incidence of double ovulation in lactating dairy cows. A secondary objective was to determine the effect of the Ovsynch@ protocol in cows with ovarian cysts. Our hypothesis was that the incidence of double ovulation in lactating dairy cows would increase as parity and milk production increased.
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MATERIALS AND METHODS Source of Data Results from the present study involved the assessmentof data collected from a previous experiment in which lactating Holstein cows underwent timed AI after synchronization of ovulation using Ovsynch@(12). Neither the objective nor the hypothesis of the present study was considered in the previous experiment. Animals and Synchronization of Ovulation Primiparous and multiparous lactating Holstein cows (n=237) from a commercial dairy herd of 600 lactating Holstein cows located in south-central Wisconsin, were blocked by parity and days in milk and were assigned randomly to 1 of 2 treatment groups for synchronization of ovulation. For both treatment groups, synchronization of ovulation was initiated at a random stage of the estrous cycle using intramuscular injections of GnRH (Cystorelin; Merial, Ltd., Iselin, NJ) and PGF2, (Lutalyse; The Pharmacia & Upjohn Co., Kalamazoo, MI) as follows: Day 0, GnRH; Day 7, PGF2,; Day 9, GnRH. Ovulation was synchronized with 25 mg PGF2, plus either 100 pg (n = 119; Group 1) or 50 pg (n = 118; Group 2) GnRH per injection. Treatment (i.e., GnRH dose) had no effect on any experimental endpoint,including the conception rate (41 vs 41.1% for the 100 pg and 50 ug treatments, respectively; 12); the double ovulation rate (13.9 vs 14.3%; 12); or the incidence of ovarian cysts (10.9 vs 11.0% for the 100 pg and 50 pg treatments, respectively). Therefore, data from cows in both treatment groups were combined to evaluate the effect of parity and milk production on the incidence of double ovulation. Although some of the cows in this herd received exogenous bST as part of the management scheme of this farm, records of bST use were unavailable and were not included in this analysis. Cows were milked 3 times daily, and the rolling herd average of the farm exceeded 10,000 kg of milk throughout the duration of the study, which was conducted as 3 replicates beginning in July and September of 1997 and in March of 1998. Each replicate was completed 66 d after initiation. Milk production (mean + SEM) of the cows used for this study averaged 40.5 f 0.8 kg/d (range = 9.1 to 72.1 kg/d) based on milk test weights recorded 2 d before AI. All cows underwent a timed AI within 12 to 18 h after the second GnRH injection. The herd manager, who remained unaware of the treatment group to which each cow was assigned, chose service sires for each mating as part of the standard reproduction program used by the farm. For each replicate, AI was performed by 4 to 5 herd personnel who also remained unaware of the treatment group to which each cow was assigned. Mean (+ SEM) days in milk of the cows at AI was 139.3 + 4 d (range = 62 to 392 d). Synchronization Rate, Double Ovulation Rate, and Ovarian Cysts Ovarian structures were monitored by using an ultrasound machine equipped with a transrectal 7.5 MHz linear-array transducer (Aloka 500V; Corometrics Medical Systems, Inc., Wallingford, CT). During each ultrasound examination, a sketch of the location and diameter of all ovarian structures 1 8 mm in diameter was recorded. For each ovarian structure, 2 diameter measurements,taken at right angles, were recorded using the digital calipers equipped with the ultrasound machine; the diameters were calculated as the mean of these 2 measurements. Ovulatory response to the second GnRH injection (synchronization rate) was determined by the
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presence of 1 or more antral follicles at the time of the second GnRH injection and the absence of 1 (single ovulation) or 2 (double ovulation) of those follicles at an ultrasound examination conducted 48 h later. Synchronization rate was calculated as the number of cows that ovulated at least 1 follicle within 48 h of the second GnRH injection, expressed as a percentage of the total number of cows receiving the Ovsynch@protocol. The double ovulation rate was calculated as the number of cows that ovulated 2 follicles within 48 h of the second GnRH injection, expressed as a percentage of synchronized cows. Fluid-filled cystic structures 2 2.5 mm in diameter identified at the scanning session conducted at the time of the second GnRH injection were classified based on ultrasonographic morphology as ovarian cysts. No attempt was made to further classify cystic structures as follicular, luteal or benign based on ultrasonographic morphology. Pregnancy Diagnosis and Conception Rate Pregnancy status was determined by using the ultrasound machine and transducer described for determining synchronization rate. Visualization of a fluid-tilled uterine horn and the presence of a fetus were used as positive indications of pregnancy. For cows in Replicate 1 (n=l 10) and Replicate 2 (n=51), pregnancy status was determined at 28 d post AI. For cows in Replicate 3 (n=76), pregnancy status was determined at 33 d post AI. Pregnancy status data at 28 and 33 d post AI for cows from all 3 replicates were combined and are reported as pregnancy status data for 28 d post AI. Conception rate at 28 d post AI was calculated as the number of cows diagnosed pregnant, expressed as a percentageof cows that ovulated at least 1 follicle within 48 h of the second GnRH injection of the synchronization of ovulation protocol. Effect of Parity and Milk Production on the Incidence of Double Ovulation To determine the effect of parity and milk production on the incidence of double ovulation, cows were classified into low (< 40 kg/d) and high (> 40 kg/d) milk production groups basedon the average milk production of 40.5 f 0.8 kg/d collected 2 d before AI. Cows were also classified by parity based on their lactation number at the time of AI (1,2 or 3+ lactations). Statistical Analysis Categorical data, which included synchronization rate, conception rate, double ovulation rate, side of ovulation, milk production group and incidence of ovarian cysts were analyzed using the Mantel-Haenszel Chi-square test (32). The linear effect of parity on incidence of double ovulation was analyzed by using maximum likelihood estimates with the logistic procedure of SAS (32). RESULTS Synchronization Rate, Double Ovulation Rate, Location of Ovulation, and Conception Rate Ovulation was synchronized in 84% (199/237) of cows receiving the Ovsynch@protocol, and the conception rate at 28 d post AI for synchronized cows was 47.6% (91/191). Of the synchronized cows, 14.1% (28/199) exhibited a double ovulation, and no cows ovulated more than 2 follicles in responseto the second G&I-I injection.
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For cows exhibiting single ovulations, the incidence of ovulation tended to be higher (FO.08) for the right than for the left ovary (Table 1). Of the cows exhibiting a double ovulation, 53.6% occurred ipsilaterally (5128 cows exhibited both ovulations on the left ovary; lo/28 cows exhibited both ovulations on the right ovary), whereas 46.4% occurred contralaterally (Table 1). Conception rate at 28 d post AI tended to be higher (~~0.08) for cows exhibiting a double ovulation than for cows exhibiting a single ovulation (Table 1). Conception rate at 28 d post AI did not differ based on the side of ovulation for cows exhibiting a single ovulation. Effect of Parity and Milk Production on the Incidence of Double Ovulation Milk production data were not available for 3 of the cows in the study that ovulated in response to the Ovsynch@protocol. One of these cows exhibited a single ovulation while the remaining 2 cows exhibited a double ovulation. Data from these 3 cows were necessarily excluded from the analyses involving milk production data. The incidence of double ovulation tended to increase linearly (PrO.09) with increasing parity; however, the incidence of double ovulation was nearly 3-fold higher (PcO.05) for cows in the high versus low milk production group (Table 2). Although statistical differences within a parity group in the proportion of cows exhibiting double ovulation in the low and high milk production groups were not detected, the incidence of double ovulation was proportionately greater within each parity group for cows in the high milk production group (Table 2). Incidence of Ovarian Cysts The overall incidence of cows classified with ovarian cysts at the time of second GnRH injection of the Ovsynch@protocol was 11% (Table 3). The incidence of ovarian cysts did not differ between cows in the low (1 l/26) and high (15/26) milk production group. There were 73.1% of cows classified as having an ovarian cyst that responded to the Ovsynch@protocol by ovulating a normal size (i.e., 10 to 22 mm in diameter) follicle other than the cystic structure. Synchronization and conception rates for cows classified as cystic did not differ from that of cows classified as normal (Table 3). In no instance did a cystic structure ovulate, and only 1 of the cows classified as cystic exhibited a double ovulation. Calving Data Calving data was collected from 58 of the 91 cows diagnosed pregnant at 28 d post AI. The remaining 33 cows either experienced subsequentembryonic loss (n=19) or were culled from the herd before they calved (n=14). Of the 58 cows that calved, 11 exhibited a double ovulation in responseto the Ovsynch@protocol; 3 of the cows that exhibited a double ovulation calved twins, whereas none of the cows exhibiting a single ovulation calved twins. Thus, the overall twinning rate of the 58 cows that calved was 5.2% (3/58). Overall gender ratio of calves was 27:34 (bulls:heifers).
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Table 1. Incidence, location of ovulation, and conception rate of lactating dairy cows exhibiting single or double ovulation after a synchronized ovulation Ovulatory response
Location of ovulation(s)
Incidence (%)
Conception rate (%)
Single ovulation (number / total)
85.9 (171/199)
Left ovary (%) 43.3” (74/171)
Right ovary (%) 56.7b (97/171)
45.2” (75/l 66)
Double ovulation (number / total)
14.1 (28/199)
Ipsilateral (%) 53.6 (15/28)
Contralateral (%) 46.4 (13/28)
64.0d (16/25)
“sbIncidenceof ovulation tended to be higher (eO.08) for the right ovary. C*dConceptionrate tended to be higher (PcO.08) for cows exhibiting double ovulation. Table 2. Effect of parity and milk production (mean f SEM) on the incidence of double ovulation after a synchronized ovulation Item Milk Production (kg/day)
Milk Production (kg/d) High (> 40) Low (< 40) 50.7 + 0.7
40.5 I!I 0.8
Parity 2 (%) (number / total)
22.2 (219) 14.3 (7/49)
10.gd (8/74)
Parity 3+ (%) (number / total)
27.8 (10/36)
20.3” (12/59)
Overall (%) (number / total)
20.2b (19/94)
13.3 (26/l 96)
Parity 1 (%) (number / total)
31.1 + 0.7
Overall
a,bProportionsdiffer by x2 analysis (KO.05). Csd,eLinear increase (FO.09) in incidence of double ovulation with increasing parity.
Theriogenology Table 3. Synchronization and conception rates after synchronization of ovulation and timed AI of lactating dairy cows classified on the basis of ovarian morphology as cystic or normal Item
Ovarian morphology” Cysticb Normal
Overall
Incidence (%)
11.0 (26/237)
89.0 (2 1l/237)
Synchronization rateC(%)
73.1 (19126)
85.3 (1801211)
84.0 (1991237)
Conception rate (%) of synchronized cows
36.8 (7/19)
48.8 (84/172)
47.6 (91/191)
“No statistical difference was detected for synchronization or conception rate using x2 analysis. bDefined morphologically by using ultrasound as a fluid-filled cystic ovarian structure 2 2.5mm in diameter present at the time of the second GnEW injection of the Ovsynch’ protocol. ‘Ovulation of a follicle by 48 hours after the second GnRH injection of the Ovsynch@protocol. DISCUSSION To our knowledge, this is the first report describing a direct relationship between milk production and the incidence of double ovulation in lactating dairy cows. Based on an epidemiological analysis of data, Kinsel et al. (19) reported that peak milk production was a major risk factor for increased twinning rates in lactating dairy cows. Because most twins in dairy cattle are dizygous (9,16,30), our results are consistent with the idea that high milk production near the time of ovulation can increase the incidence of double ovulation and that this increase may subsequently result in increased twinning. The practical implication of this relationship is important, because current dairy management strategies aim to maximize milk production per cow, thereby increasing the frequency of double ovulation in the dairy cattle population as a whole. In support of this notion, the overall twinning rates reported for dairy cows in recent studies are higher than those reported previously (6). If twinning is related to milk production, this increase should not be unexpected considering the annual increases in milk production per cow that have occurred over this time. Overall, the incidence of double ovulation after a synchronized ovulation in the present study was 14.1%. Although few recent studies have reported the incidence of double ovulation, previous reports in which palpation per rectum was used to determine the number of CL present 7 to 13 d after a spontaneousestrus reported double ovulation rates of 13.1% (18) and 5.4% (20). However, becausethere was considerable disparity in the incidence of double ovulation between these studies, the technical ability to detect multiple CL using palpation per rectum, particularly in the earlier study, needs to be questioned. In our present study, we used transrectal ultrasonography to determine synchronization and double ovulation rates, a technique with a high degree of accuracy for resolving ovarian structures and events such as ovulation in cattle (15). Although we do not know whether the Ovsynch@protocol directly affected the incidence of
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double ovulation in the present study, the incidence of multiple CL before initiation of ang hormonal treatment was similar to the incidence of double ovulation in response to Ovsynch (15 vs 13%, respectively; n=156; our unpublished data). In addition, the observation that the double ovulation rate is related to milk production also argues that this methodology provides relevant information on factors affecting double ovulation. Twinning rates in dairy cattle are dramatically affected by parity, with an incidence of less than 1% for virgin heifers but approaching nearly 10% for older lactating cows (4,21,30). Although the effect of parity on twinning is riot clearly understood, increased uterine capacity of cows calving twins has been implicated (30). The incidence of double ovulation, as with the incidence of twinning, has also been reported to increase with parity in lactating dairy cows, increasing from 2.9% for Parity 1 to 5.9% for Parity 3. (20). Although our results demonstratea linear increase in the incidence of double ovulation with increasing parity, the apparent reason for this increase was that the proportion of cows with high milk production within a parity group was greater for the older cows. Thus, the physiology of high milk production in and of itself appearsto increase the incidence of double ovulation independent of either age or parity. Specific physiologic mechanisms that may predispose higher producing dairy cows to exhibit more double ovulations are not known. Increased amounts of dietary bypass protein can increase the ovulation rate and the incidence of twinning in ewes (26). High levels of bypass protein fed to lactating dairy cows may increase the incidence of double ovulation and hence the rate of twinning similar to that of the practice of “flushing” in ewes (7). Unilateral ovariectomy increased the incidence of double ovulation on the remaining ovary in dry dairy cows and pubertal dairy heifers without affecting the number of follicular waves per cycle (23). Despite this observation, FSH, LH and progesterone concentrations before and after unilateral ovariectomy did not differ, suggesting that the magnitude of endocrine changes required for 2 follicles to undergo deviation during a follicular wave may be too small to measureor may be of a short duration (23). Our recent data (35) suggest that high feed intake in lactating dairy cows may increase hepatic metabolism of ovarian steroids, similar to that reported previously in ewes (22,27). These changes in steroid metabolism may alter the endocrine environment sufficiently to allow for deviation of 2 follicles during the selection period of a follicular wave. Future studies may provide further insight into the physiological mechanisms that mediate the effects of high milk production on double ovulation rate. Although double ovulation must precede the occurrence of dizygous twins, double ovulation does not consistently result in twinning becausecows carrying twins experience high rates of embryonic loss and abortion during gestation (6). However, there were too few calvings in the present study to reliably assesspregnancy attrition in cows exhibiting double ovulation. The analysis of double ovulation rather than twinning allowed us to directly observe a physiologic event that may be affected by milk production without the confounding problems of poor fertility (11) and the potentially higher pregnancy losses (34) that occur in high producing dairy cows. Thorough analysis of the events that occur between double ovulation and twin births will be critical for understanding the factors regulating twinning in dairy cattle. A secondary objective of the present study was to evaluate the effect of Ovsynch’ on cows with ovarian cysts. The incidence of cows classified as cystic in the present study was ll%,
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similar to the reported rates of 10 to 13% in lactating dairy cows (2,lO) but less than that of problem herds, which may experience incidences of 30 to 40% over brief periods (1). Ovarian cysts in cattle commonly are defined as anovular fluid-filled structures ~25 mm in diameter that persist on the ovaries for more than 10 d (1). Because we evaluated cysts only at the time of the second GnRH injection, it was not possible to physiologically classify the cysts or assessthe their response to the first or second injection of the Ovsynch@ protocol. The physiology and etiology of ovarian cysts is poorly understood, and, although milk production has been implicated (17), it did not affect the incidence of ovarian cysts in our present study. Current hormonal therapies for ovarian cysts vary depending on the classification of the cyst. Although follicular cysts are most commonly treated with G&I-I (5,33,36), the observation that nearly 20% of cows with follicular cysts recover spontaneously (5) supports the notion that many ovarian cysts are benign and, therefore, do not interfere with the normal progression of follicular waves. Treatment of a follicular cyst with GnRH induces luteinization rather than ovulation and results in formation of a luteal cyst (13). Once formed, regression of luteal cysts can be induced by administration of PGFra (24). Other researchers (1,13) have reported the similar observation that cows with ovarian cysts frequently ovulate a normally growing follicle in response to GnRH treatment. Data from the present study also indicate that synchronization and conception rates of cows classified as cystic did not differ statistically from that of cows classified as normal. Collectively, these data support the use of Ovsynch’ as a treatment for lactating dairy cows exhibiting ovarian cysts. We conclude that milk production is the primary factor affecting the incidence of double ovulation in lactating dairy cows. Further experiments are needed to elucidate specific mechanisms whereby the physiology of increased milk production affects the number of follicles that undergo deviation and acquire ovulatory capacity during a follicular wave. Future strategies may then be developed to avoid or reduce the negative effects of twinning on dairy operations. In addition, the Ovsynch@ protocol may be an effective method for establishing pregnancy in lactating dairy cows with ovarian cysts REFERENCES 1. Archibald LF, Thatcher WW. Ovarian follicular dynamics and management of ovarian cysts. In: Van Horn HH, Wilcox CJ (eds), Large Dairy Herd Management. Champaign IL: Am Dairy Sci Assoc, 1992;199-208. 2. Bartlett PC, Ngategize PK, Kaneene JB, Kirk JH, Anderson SM, Mather EC. Cystic follicular disease in Michigan Holstein-Friesian cattle: incidence, descriptive epidemiology, and economic impact. Prev Vet Med 1986;4:15-33. 3. Beerepoot GMM, Dykhuizen AA, Mielen M, Schukken YH. The economics of naturally occurring twinning in dairy cattle. J Dairy Sci 1992;75:1044-1051. 4. Berry SL, Ahmadi A, Thurmond MC. Periparturient disease on large, dry lot dairies: interrelationships of lactation, dystocia, calf number, calf mortality, and calf sex. J Dairy Sci 1994;77(Suppl 1):379 abstr. 5. Bierschwal CJ, Garverick HA, Martin CE, Youngquist RS, Cantley TC, Brown MD. Clinical responseof dairy cows with ovarian cysts to GnRH. J Anim Sci 1975;41:1660-1665.
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6. Day JD, Weaver LD, Franti CE. Twin pregnancy diagnosis in Holstein cows: Discriminatory powers and accuracy of diagnosis by transrectal palpation and outcome of twin pregnancies. Can Vet J 1995;36:93-97. 7. Dunn TG, Moss GE. Effects of nutrient deficiencies and excesseson reproductive efficiency of livestock. J Anim Sci 1992;70:1580-1593. 8. Eddy RG, Davies 0, David C. An economic assessmentof twin births in British dairy herds. Vet Ret 1991;129:526-529. 9. Erb RE, Morrison RA. Effects of twinning on reproductive efficiency in a Holstein-Fresian herd. J Dairy Sci 1959;42:512-519. 10. Erb RE, White ME. Incidence rates of cystic follicles in Holstein cows according to 15-day and 30-day intervals. Cornell Vet 1981;71:326. 11. Faust MA, McDaniel BT, Robinson OW, Britt JH. Environmental and yield effects on reproduction in primiparous Holsteins. J Dairy Sci 1988;71:3092-3099. 12. Fricke PM, Guenther JN, Wiltbank MC. Efficacy of decreasing the dose of GnRH used in a protocol for synchronization of ovulation and timed AI in lactating dairy cows. Theriogenology 1998;50:1275-1284. 13. Garverick HA. Ovarian follicular cysts in dairy cows. J Dairy Sci 1997;80:995-1004. 14. Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Minireview. Selection of the dominant follicle in cattle. Biol Reprod 1996;55:1187-l 194. 15. Griffin PG, Ginther OJ. Researchapplications of ultrasonic imaging in reproductive biology. J Anim Sci 1992;70:953-72. 16. JohanssenI, Lindhe B, Pirchner F. Causesof variation in the frequency of monozygous and dizygous twinning in various breedsof cattle. Hereditas 1974;78:201-234. 17. Johnson AD, Legates JE, Ulberg LC. Relationship between follicular cysts and milk production in dairy cattle. J Dairy Sci 1966;49:865-868. 18. Kidder HE, Barrett GR, Casida LE. A study of ovulations in six families of HolsteinFriesians. J Dairy Sci 1952;35:436-444. 19. Kinsel ML, Marsh WE, Ruegg PL, Etherington WG. Risk factors for twinning. J Dairy Sci 1998;81:989-993. 20. Labhsetwar AP, Tyler WJ, Casida LE. Analysis of variation in some factors affecting multiple ovulations in Holstein cattle. J Dairy Sci 1963;46:840-842. 21. Marcusfeld 0. Periparturient traits in seven high dairy herds, Incidence rates, association with parity, and interrelationships among traits. J Dairy Sci 1987;70:158-166. 22. McEvoy TG, Robinson JJ, Aitken RP, Findlay PA, Palmer RM, Robertson IS. Dietaryinduced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova. Anim Reprod Sci 1995;39:89-107. 23. Mohan M, Rajamahendran R. Effects of unilateral ovariectomy on follicular development and ovulation in cattle. Theriogenology 1998;49:1059-1070. 24. Nanda AS, Ward WR, Dobson H. Retrospective analysis of the efficacy of different hormone treatments of cystic ovarian diseasein cattle. Vet Ret 1988;122:155. 25. Nielen M, Schukken YH, Scholl DT, Wilbrink HJ, Brand A. Twinning in dairy cattle: a study of risk factors and effects. Theriogenology 1989;32:845-862. 26. Nottle MB, Hynd PI, SeamarkRF, Setschell BP. Increasesin ovulation rate in lupin-fed ewes are initiated by increasesin protein digested postruminally. J Reprod Fertil 1988;84:563-566.
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27. Parr RA, Davis IF, Miles MA, Squires TJ. Feed intake affects metabolic clearance rate of progesterone in sheep.Res Vet Sci 1993;55:306-310. 28. Pfau KO, Bartlett JW, Shuart CE. A study of multiple births in a Holstein-Friesian herd. J Dairy Sci 1948;31:241-254. 29. Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGFzuand GnRH. Theriogenology 1995;44:915-923. 30. Ryan DP, Boland MP. Frequency of twin births among Holstein-Friesian cows in a warm dry climate. Theriogenology 1991;36:1- 10. 3 1. Sat-tori R, Fricke PM, Ferreira JCP, Ginther OJ, Wiltbank MC. Acquisition of ovulatory capacity by ovarian follicles during growth of follicular waves in lactating dairy cows. J Dairy Sci 1998;81(Suppl 1):223 abstr. 32. SAS Institute Inc., SAS/STAT User’s Guide, version 6. Cary NC: SAS Institute Inc, 1989. 33. Seguin BE, Convey EM, Oxender WD. Effect of gonadotropin-releasing hormone and human chorionic gonadotropin on cows with ovarian follicular cysts. Am J Vet Res 1976;37:153-157. 34. Vasconcelos JLM, Silcox RW, Lacerda JA, Pursley JR, Wiltbank MC. Pregnancy rate, pregnancy loss, and response to heat stress after AI at 2 different times from ovulation in dairy cows. Biol Reprod 1997;56(Suppl 1):140 abstr. 35. Vasconcelos JLM, Bungert KA, Tsai SJ, Wechsler FS, Wiltbank MC. Acute reduction in serum progesterone concentrations due to feed intake in pregnant lactating dairy cows. J Dairy Sci 1998;81(Suppl 1):226 abstr. 36. Whitmore HL, Hurtgen JP, Mather EC, Seguin BE. Clinical response of dairy cattle with ovarian cysts to single or repeated treatments of gonadotropin-releasing hormone. JAVMA 1979;174:1113-1115.