Negative effects of anovulation on reproductive performance of cattle have been reported during all of the last century. Detailed descriptions of cystic cows and anovular cows with small ovaries can be found in the literature from the early 1900s. In an early report, Hancock (1948) cleverly proposed that cows could be classified according to the type of ovarian activity into three main categories: ovulatory cows, anovulatory cows (cystic or non-cystic), and cows with inactive ovaries. Unfortunately, in these early studies the understanding of the causes leading to the anovular condition was limited; therefore, the development of effective treatments was restricted. Today, with the help of technologies such as ultrasonography and hormone assays, we understand more about the dynamics of follicular growth and circulating reproductive hormones. This has allowed for a better evaluation of the potential underlying causes leading to anovulation as well as the development of treatments to resolve this condition.
The term anovular is used to identify a cow that is not ovulating at or near the time that breeding of these cows should occur. This can be accurately determined by sequential ultrasonographic exams or measurements of progesterone (P4) concentration in blood. However, in some field situations where the estimation of cyclicity depends on occasional palpations per rectum and the detection of estrus, cows that are not detected in estrus are, sometimes, assumed to be anovular. Unfortunately, there are many physiological and environmental reasons why a cow may not display signs of estrus even though the cow has an LH surge and ovulates. For example, one reason might be an increased level of milk production. Recently we have conducted a study that used a system that allows continuous estrous detection (HeatWatch, DDX Technologies) to evaluate the effect of milk production on expression of estrus. We found that cows with high milk production have a much shorter duration of standing estrus (Lopez et al., 2004). In addition, a greater percentage of the higher producers had silent ovulations (ovulations with no standing estrus events) than were found in lower producers. Regardless of the different physiological reasons for the antagonistic association between high milk production and expression of estrus, this produces a situation in which higher producers are less likely to be found in estrus and might be misdiagnosed as anovular cows. Alternatively, some cows that are not ovulating may be detected in estrus, either inaccurately or due to expression of estrus in the absence of ovulation. Thus, designation of a cow as anovular (lack of ovulation) based on circulating P4 concentrations or multiple ultrasound evaluations of the ovary may identify a different group of cows then might be designated as anestrus (no estrus detected) in a particular commercial dairy operation. In this manuscript we will use the term anovular to identify cows that are not ovulating and only report results of studies that have objectively determined this status by multiple measurements of circulating P4 concentrations and/or multiple evaluations of the ovary by ultrasound.
Incidence of Anovulation
Studies in dairy cows that have evaluated cyclicity by P4 concentrations in sequential blood samples taken 10 to 12 d apart have reported an incidence of anovulation ranging from 15 to 54 % by 49 to 71 days postpartum (Table 1). In two studies that we conducted, weekly ultrasound exams were combined with P4 concentrations to evaluate cyclicity. We observed incidences of anovulation of 20.2 % (64 of 316 cows; Gümen et al., 2003) and 28.5 % (76 of 267 cows) by 60 and 71 days in milk (DIM), respectively. In addition, primiparous cows generally have a higher incidence of anovulation than multiparous cows. Thus, the incidence of anovulation in modern dairy cows is high and represents a major concern for producers.
|Moreira et al., 2001||37.3%||15.7%||n = 499 @ 63 DIM by P4|
|Gumen et al., 2003||28% (34/122)||15% (29/194)||@ 57 DIM by ultrasound & P4|
|Lopez et al., 2005||29% (38/131)||27.9% (38/136)||@ 71 DIM by ultrasound & P4|
|Chebel et al., 2006||54.1% (210/388)||31.5% (183/580)||@ 49 DIM by P4|
Causes of Anovulation
We will discuss the causes of anovulation from two different perspectives. First, we will discuss the underlying physiology that causes cows to not ovulate. Second, we will discuss the on-farm or epidemiological factors that have been associated with anovulation.
Physiology Underlying Anovulation
We have previously reviewed the different physiological states that can result in anovulation in dairy cattle (Wiltbank et al., 2002). The most obvious cause for lack of ovulation is the presence of a persistent corpus luteum on the ovary. Obviously, the presence of a persistent corpus luteum and lack of ovulation is associated with pregnancy, and this would not be considered a problem of reproductive efficiency. In addition, some non-pregnant cows (1.5 to 6.4 %) will also have a persistent corpus luteum that does not regress during a 25 day time (reviewed in Wiltbank et al., 2002). The incidence of a persistent corpus luteum increases dramatically in cows that ovulate during the first 25 days after calving (25 %) compared to cows ovulating later (0 %) (Ball and McEwan, 1998). This problem can be effectively treated by regressing the persistent corpus luteum using prostaglandin (PG) F2α. This cause of anovulation will not be discussed further and will not be considered in any of our subsequent discussions on incidence or treatment of anovulation.
There are three primary follicular growth patterns that have been observed in anovular dairy cows (for review and references see Wiltbank et al., 2002). The first pattern represents the classical anestrous or anovular cow with growth of relatively small follicles. In this pattern, follicles only reach a maximal size of 9-15 mm in diameter. This pattern is generally associated with lower body condition score and negative energy balance. The underlying physiology causing this pattern appears to be a deficiency of LH pulses. The low energy status of the cow appears to cause the inadequate number of LH pulses and this causes insufficient follicle growth and insufficient production of estradiol by the follicle. Thus, these cows never have a follicle that reaches sufficient size or estradiol production to induce the cow to come into estrus and have an LH surge and ovulate.
This problem of inadequate follicle growth (small follicles) is more prevalent during the early postpartum period when cows undergo the lowest negative energy balance. As cows reach an improved energy and nutrient status, LH pulses will increase and follicles will grow to sufficient size resulting in estradiol production to cause the animal to come into estrus and ovulate (Beam and Butler, 1999; McDougall et al., 1995). This problem is most associated with cows having low body condition score (2.5 or lower).
A second follicle growth pattern that is readily identified by veterinarians is growth of follicular cysts (follicles > 25 mm in diameter). In most cases these cows do not ovulate or show behavioral estrus, in spite of the presence of high circulating estradiol concentrations in these cows. This problem is due to a lack of estradiol positive feedback at the hypothalamus (Nanda et al., 1991; Gumen and Wiltbank, 2002). In other words, the cystic cow is resistant to the effects of estradiol on the brain. She does not show estrus and does not have an LH surge or ovulate.
The third pattern of follicular growth in anovular cows is the most prevalent pattern that we have found in our studies of lactating dairy cows (Gumen et al., 2003; Lopez et al., 2005), but also is the most difficult to diagnose in commercial dairies. In cows with this type of anovulation, follicles grow to ovulatory size (16-24 mm) but do not ovulate. The ovary does not have structures that would be readily identified as cystic structures, but appears to be in a similar physiological state. In other words, these cows appear to have high circulating estradiol but do not show standing estrus or ovulation. Some of the secondary signs of estrus (mucus, activity) may be present in many of these cows on a consistent basis even though these cows remain anovular.
There may be different problems that cause these three different types of anovulation. At this time most of the studies that have been done have not clearly designated the anovular cows into the physiologic state that underlies the lack of ovulation. It seems very likely that cows in each of these three categories will respond differently to different treatments. Nevertheless, because of inadequate information all three of these types of anovular cows will be grouped together in subsequent discussions.
On-Farm Factors Associated with Anovulation
This section discusses several risk factors that have been found to influence the incidence of anovulation on dairy operations. Although numerous studies on anovulation could be cited, we will emphasize a few recent studies that have been done on anovulation.
The relationship between milk production and anovulation has been examined with inconsistent findings. Erb (1984) reviewed four North American studies in which they contrasted the levels of milk production or genetic potential for milk production between cows that later became anovulatory and cows that did not. They concluded that high milk production did not cause anovulation in Holstein dairy cows, but anovulatory cows produced more milk than their herdmates (Erb, 1984). Bartlett et al. (1986) compared milk production between anovulatory (n=338) and ovulatory lactations (n=2262). They reported that lactations with anovulatory follicles produced an average of 422 more kg of 305 mature equivalent (ME) milk than lactations without anovulatory follicles. Thus, these studies support the argument that high milk production does not cause anovulation, yet anovulatory follicles may be related to increased milk production. In one of our recent studies on anovulation we have evaluated the relationship between milk production from 60-70 DIM and the incidence of anovulation at 71 DIM (Lopez et al., 2005). Regardless of the level of milk production, about 28 % of cows were found to be anovular. Thus, in contrast to duration of estrus, which is closely related to the level of milk production (Lopez et al., 2004), lack of ovulation appears to be due to causes other than level of milk production. It should be remembered that these data were collected from a high-producing dairy herd fed a well-balanced, total mixed ration. In herds that are limiting for energy or other nutrients (grazing dairies, not well-balanced rations), it seems very likely that higher milk producers would have a much greater incidence of anovulation than low producers. Similarly, a recent study from California (Chebel et al., 2006) reported that the percentage of cows that were anovular at 49 days postpartum was unrelated to milk production (P = 0.61) with cows in the lowest (34.2 kg/d) or highest (51.3 kg/d) quartiles for milk production having similar anovulation rates (42.6 % vs. 43.1 %). Thus, in high-producing, well-fed dairy herds it seems that incidence of anovulation is probably primarily related to factors other than level of milk production.
Body Condition Score
Body condition score (BCS) is an arbitrary scale for estimating the amount of body fat in cows (Wildman et al., 1982). Incidence of anovulation and BCS around 60 days postpartum are strongly correlated (Cartmill et al., 2001; Moreira et al., 2001; Gumen et al., 2003; Lopez et al., 2005). For example, percentage of cows found to be anovular at 71 days postpartum (Lopez et al., 2005) decreased as BCS increased from 83.3 % (BCS < 2.5), 38.2 % (2.5), 34.4 % (2.75), 21.8 % (3.0), to 8.5 % (> 3.25). Similarly Chebel et al., 2006 reported that percentage anovular at 49 days postpartum was related to BCS with cows having BCS < 2.75 being 45.2 % anovular and cows > 2.75 being 33.7 % anovular. Thus, there is a relationship between percentage anovular and BCS.
It should be noted that from 44 % (Chebel et al., 2006) to 63 % (Lopez et al., 2005) of anovular cows have been found to have good BCS. Thus, BCS does not completely explain the high incidence of anovulation in dairy cows. In addition, Chebel et al. (2006) reported that change in BCS from 3 to 62 DIM was not related to incidence of anovulation (P = 0.20). It seems likely that many other on-farm factors besides BCS and negative energy balance are causing the high rates of anovulation observed in lactating dairy cows, particularly the type of anovulation with larger follicles (categories 2 and 3 described above). Although cows with low BCS have a higher incidence of anovulation, many cows with anovulation do not have low BCS.
Most studies have shown a greater incidence of anovulation in primiparous than multiparous cows (see Table 1). For example, Moreira et al (2001) determined anovulation by measuring low circulating P4 concentrations in two blood samples and reported a 37.3 % incidence of anovulation in primiparous cows and only a 15.7 % incidence in multiparous cows. In one of our studies, we found that primiparous cows (28 %) had higher incidence of anovulatory condition (anovulatory cows were determined by ultrasonography plus circulating P4 concentration) than multiparous cows (15 %) in one commercial dairy herd. The higher incidence of anovulation in primiparous cows occurred at all levels of BCS, suggesting that this increased incidence may not be due to only a decreased BCS in primiparous cows. Chebel et al. (2006) also reported that primiparous cows (54.1 %) had higher rates of anovulation at 49 days postpartum than multiparous cows (31.5 %). Nevertheless, the relationship between anovulation and age is somewhat controversial and appears to vary by herd. There could be numerous reasons that explain the variability in whether age of the cow affects the incidence of anovulation including: competition for resources between older and younger cows, nutritional management, production level, early postpartum care differences, and methods used to determine anovulation in different herds or studies.
Summary of Risk Factors
In addition to the above factors, uterine infections, feeding monensin, and phytoestrogens in feed have been reported to be risk factors for anovulation in some studies. Thus, it seems clear that there are multiple possible mechanisms that could underlie anovulation in dairy herds. In herds with low BCS, it seems likely that negative energy balance is the underlying basis for anovulation. Any management changes that result in better BCS near the start of breeding should decrease the incidence of anovulation. However, it seems clear that in well-fed dairy cows with good BCS there is still a surprisingly high incidence of anovulation. This type of anovulation is characterized by large follicles and, most likely, resistance to the positive feedback effects of estradiol. The next two sections will present some brief ideas on potential methods to prevent anovulation in lactating dairy cows, as well as hormonal methods to treat anovulation.
Prevention of Anovulation by Non-Hormonal Treatments
Based on the risk factors discussed above, there are a number of procedures that could be implemented to decrease anovulation at the start of the breeding period in dairy cattle.
The most discussed method for reducing anovulation is improving nutritional status of the cow, which has been extensively discussed in previous manuscripts (Beam and Butler, 1999; Ferguson, 1996).
The second most obvious method for reducing anovulation at the start of the breeding period is to delay the time of first breeding. We have previously reported (Lopez et al., 2005) that 53.9 % of anovular cows at 71 days postpartum will spontaneously recover by 100 days postpartum. Thus, the earlier the start of the breeding period, the greater the percentage of anovular cows present in the breeding group. Indeed, Tenhagen et al. (2003) showed a clear improvement in first service conception rate when timed AI was delayed from 53-59 DIM (14.4 %), to 73-81 DIM (28.7 %), and up to 94-101 DIM (41.4 %). This improvement in conception rates is likely due to a reduction in anovular cows, as well as other improvements in reproductive function with increasing days after calving.
Another method we have found to decrease anovulation is to reduce the length of the dry period (Gumen et al., 2005a; Watters et al., 2006). The early time to first ovulation appears to be due to a reduction in negative energy balance in cows with shortened dry period lengths (Gumen et al., 2005a). The days to first ovulation was reduced from 43 to 35 days and the percentage of cows that were anovular at 70 DIM declined from 18 % to 8 % when the dry period was shortened from 56 to 34 days (Watters et al., 2006). It should be noted that cows with the reduced dry period lengths were placed immediately on the moderate energy diet (steam-up ration) at the time of initial dry off and were never given the typical low energy, dry-cow ration. This protocol was particularly advantageous for older cows (cows going into the dry period after their second or greater lactation) with days open reduced from 133 days on the traditional dry period to 113 days on the shortened dry period. This improvement was not due to reduced milk production as older cows had similar fat-corrected milk production whether they were on the traditional or shortened dry period.
There are likely to be a number of non-hormonal methods that can reduce the incidence of anovulation in lactating dairy cows. It seems clear that better nutritional programs, particularly programs that reduce the amount of negative energy balance, will help reduce the incidence of anovulation in dairy cows. In addition, delaying the beginning of the breeding period, perhaps combined with reducing the dry period length, particularly in older cows, should help reduce anovulation. In spite of these important prevention programs, it seems clear that hormonal protocols also need to be implemented to assure that anovular cows have every opportunity to become pregnant during their lactation.
Hormonal Treatments of Anovular Cow
Prior to 1943, treatment of follicular cysts or anovulation in cows was mostly limited to either uterine irrigation with physiological salt solution or manual rupture of the anovulatory follicles. Casida et al. (1944) indicated that treatment with pituitary extracts containing gonadotropins could also be an effective therapy for follicular cysts. Following results of Casida et al. (1944) many hormones (GnRH, human chorionic gonadotropin, P4, or combination of these hormones) have been used to treat anovulation.
Most economic analyses have found that anovular cows can reduce dairy herd profitability due to increased risk of culling and increased inseminations per conception (De Vries et al., 2006). Thus, effective treatment of anovular cows, in a timely manner, is likely to improve reproductive efficiency in a commercial dairy herd and may increase dairy farm profitability.
The most utilized treatment for anovular cows in the U.S.A. is the Ovsynch protocol. This protocol utilizes GnRH, followed 7 days later with PGF2α, and 48-56 hours later with a second GnRH, and a timed AI at 14-18 hours after the second GnRH treatment. This protocol appears to induce ovulation in a high percentage of anovular dairy cows, but some of these cows have a subsequent short luteal phase (Gumen et al., 2003). Table 2 shows some of the recent studiesthat have compared ovular and anovular cows treated with Ovsynch. In almost every study there were significantly lower conception rates in non-cycling than cycling cows treated with Ovsynch. Thus, although Ovsynch may induce ovulation in non-cycling cows, there is still likely to be a reduction in conception rates in these cows. It seems likely that the later the DIM at the start of Ovsynch, the more successful it will be in cycling or non-cycling cows.
Treatment with P4 has been effectively used to induce cyclicity in anovular cows for many years. In recent years a number of groups have combined the use of a P4 releasing device (CIDR, controlled internal drug releasing, Pfizer Animal Health) with the Ovsynch timed AI protocol into a protocol termed CIDR-Synch. In this protocol, cows have the insert placed in the vagina at the same time as the first injection of GnRH in the Ovsynch protocol and the CIDR insert is removed at the time of PGF2α treatment. The use of the CIDR-Synch protocol compared to the Ovsynch protocol has recently been reviewed (Stevenson et al., 2006). The results are surprisingly variable and no clear advantage can be scientifically espoused at this time.
|Study||Percentage Pregnant to First AI||Comments|
|Moreira et al., 2001||41.7% (159/382)||22.4% (26/117)||Ovsynch TAI @ 73 DIM|
|Cordoba & Fricke, 2001||45.8% (33/72)||30.0% (9/30)||Grazing Dairy; TAI @ 66 DIM|
|Gumen et al., 2004||32% (37/117)||9% (3/33)||Ovsynch TAI @ 64-70 DIM
Anovular @ 54-60 DIM
|Galvao et al., 2004||35.4% (91/257)||22.9% (14/61)||Heatsynch TAI @~65 DIM|
|Chebel et al., 2006||31.1% (178/573)||20.9% (82/393)||Anovular @ 49 DIM
Some AI to estrus
Ovsynch TAI @ 72 days
Presynch with CIDR in some cows
|Stevenson et al., 2006||34.7% (78/225)||30.2% (29/96)||Ovsynch TAI @ 89 ± 42 DIM|
A strategy that is currently being investigated is the use of presynchronization treatments that are designed to cause ovulation in non-cycling cows. The classical Presynch protocol involved two injections of PGF2α at a 14 day interval. This is followed 12-14 days later with the first GnRH of the Ovsynch protocol. This protocol has been shown to improve conception rates in cycling dairy cows (Moreira et al., 2001). However, treatments with PGF2α are unlikely to improve reproduction in non-cycling dairy cows and this appears to be the case in previous studies (Moreira et al., 2001). Recently we have completed a study using a GnRH treatment 7 days prior to the final PGF2α treatment of a Presynch protocol (Gumen et al., 2005b). This protocol was found to induce ovulation and increase P4 concentrations at the time of the second PGF2α treatment of the Presynch protocol. However, we did not find an improvement in conception rates using this protocol (Gumen et al., 2005b). Recently, Chebel et al. (2006) has reported using a CIDR insert for the 7 days prior to the final PGF2α of the Presynch protocol. This treatment induced cyclicity, as evidenced by an increase from 30 % of control anovular cows (at 49 DIM) initiating cyclicity by 62 DIM to ~47 % of CIDR-treated cows initiated cyclicity. Nevertheless, presynchronization with the CIDR was not found to improve conception rate at the first AI.
A recent study by Bello et al. (2006) utilized a novel presynchronization protocol with PGF2α and GnRH. The protocol utilized a PGF2α treatment followed 2 days later by GnRH and followed 6 days later by the first GnRH of the Ovsynch protocol. This treatment appeared to produce an excellent presynchronization of cycling cows and theoretically may help non-cycling cows, but it has not yet been tested in a large fertility study. Thus, presynchronization treatments that will allow initiation of the Ovsynch protocol on the correct days of the estrous cycle as well as induce cyclicity in anovular cows are clearly possible. Future studies are needed to optimize these presynchronization protocols so that improvements in reproductive performance can be produced.
A number of other hormonal treatments for anovular cows during the synchronization protocol have been utilized in numerous other studies. These protocols utilize treatments with estradiol during the synchronization protocol. Although the results of these studies are intriguing and encouraging, estradiol is not currently approved for use in lactating dairy cows in the U.S.A. Therefore, these protocols will not be reviewed in this manuscript.
There is a great deal of information on anovular cows that has become available in the last 10 years due to the use of ultrasonography to evaluate the growth patterns of anovular follicles and to accurately diagnose the incidence and response to treatment of anovular cows. It is clear that dairy cows with low BCS, negative energy balance, and smaller follicles display one of the important types of anovulation; however, a large number of anovular cows do not fit this classical idea. These anovular cows have large ovulatory-sized follicles and normal BCS. Reductions in percentage of anovular cows can be produced by well-designed nutritional programs, delayed time to first breeding, and reduced dry period length. These prevention methods can reduce the impact of anovular cows in reproductive management programs. In addition, hormonal programs are effective in inducing cyclicity in anovular cows. Treatment with Ovsynch induces ovulation in many anovular cows in response to the GnRH treatments used in the protocol. Most studies suggest that Ovsynch will increase the number of pregnancies in anovular cows, even though there are reductions in the conception rates for anovular compared to ovular cows that are treated with Ovsynch. Other hormonal treatment protocols using novel Presynch protocols and CIDR-Synch are still being tested and optimized at this time.
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Milo C. Wiltbanka, Ahmet Gumenb, Hernando Lopezc, and Roberto Sartorid
aDepartment of Dairy Science, 1675 Observatory Drive, University of Wisconsin-Madison, Madison, WI 53706 – Wiltbank@wisc.edu
bDepartment of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey – Agumen@uludag.edu.tr
cABS Global, 1525 River Road, DeForest WI 5353 – HLopez@absglobal.com
dEmbrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil – Sartori@cenargen.embrapa.br