Title	:	The Future of Dairy Reproduction
Source	:	Texas A&M University
Author	:	Thatcher
Date	:
Content	:

The Future of Dairy Reproduction

W.W. Thatcher1, F. Moreira1, M. Pancarci1, E.R. Jordan3 and C.A. Risco2 1Department of Animal Sciences, IFAS and 2Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, 32611-0920, and 3Department of Animal Science, Texas A&M University, Dallas, 7252-6599, TX.

Introduction

The goal of maintaining herd pregnancy rates in current production systems is a challenge due to large herds, confinement systems that are not necessarily conducive to heat detection, cow identification, and the challenge of implementing nutritional management systems that meet individual cow requirements during both the transition and lactation periods that ultimately impact reproductive performance. Reproductive management is further compromised by the seasonal period of heat stress that reduces both heat detection rates and pregnancy rates to inseminations at detected heats. Under our current production systems, the heat detection rate in high-producing cows is 50% at best.

The goal for a successful estrus synchronization program is precise control of estrus, which will allow fixed-time AI without the need for estrus detection. However, this needs to be coupled with high fertility at the synchronized estrus or ovulation. The target populations are dairy cattle, in which herd pregnancy rates are often times low due to poor heat detection, low conception rates and occurrence of anestrus, and beef cattle that have a high incidence of anestrus at the designated time of breeding. Strategies for ovulation control have been based on controlling life span of the CL with prostaglandins, induction of ovulation with GnRH, or prevention of estrus using progestagen treatments. As our knowledge regarding control of the estrous cycle has been expanded, appropriate combinations of these approaches that are physiological have been successful. Prostaglandins alone do not provide acceptable synchrony, because the time of ovulation depends on the stage of development of the dominant follicle at the time of prostaglandin-induced regression of the CL. This problem has been resolved partially with the injection of GnRH that causes ovulation of the dominant follicle at certain stages of the cycle and recruitment of a new dominant follicle. Prostaglandin is injected 7 days after GnRH to induce CL regression at a time a new dominant follicle is present that induces estrus 48 to 72 h later. Thus this system has synchronized follicle development with CL regression. This system can be expanded further with a second GnRH injection given 48 h after prostaglandin injection to synchronize precisely the time of ovulation of the dominant follicle. Precision of follicle recruitment following the first injection of GnRH is dependent upon successfully turning over a dominant follicle. Thus the concept of pre-synchronization was introduced to enhance the probability of having a dominant follicle (> 10mm) that will be induced to ovulate to the first GnRH injection and the assurance that a CL will be present throughout the synchronization period (i.e., a CL will not regress prior to the time that prostaglandin is injected).

Progestogens have been utilized in several ways as part of a synchronization program. Long-term treatment with a progestogen (e.g, 14 days) permits spontaneous CL regression to occur followed by withdrawal of the progestogen to allow a synchronized estrus. Such programs resulted in excellent synchrony of estrus but poor conception or pregnancy rates because of aged-persistent follicles of low fertility. However, progestogen treatments could be used to pre-synchronize estrus and allow for subsequent implementation of a synchronization program that would benefit from the staged presence of a new dominant follicle and CL. Alternatively, progestogen treatment could be given concurrently with GnRH and PG treatments to insure progestogen exposure, prevent premature occurrence of estrus, and enhance cyclic responses in anestrous animals. At the present time, MGA, an oral progestin, has recently received clearance from FDA for use in reproductive classes of beef and dairy females. No other progestin (implant [e.g., Norgestomet] or intravaginal insert [CIDR or PRID] is approved currently (July, 2001) for use in the USA.

Estradiol benzoate and estradiol valerate have been used to induce follicle turnover with or without injection of progesterone. Injection of the estrogens reduces FSH secretion such that FSH dependent follicles (follicles undergoing recruitment e.g., 2 to 8 mm) will undergo regression, whereas the injection of progesterone will induce turnover of LH–dependent follicles (e.g., > 10 mm). Thus, estrogens and progestogens given concurrently will induce follicle turnover and new wave emergence regardless of what stage of the follicular wave and/or stage of the estrous cycle. In contrast, GnRH will only induce follicle turnover in follicles > 10 mm. However, these estrogens are not approved in the USA for cattle. An alternative use of estrogens is their ability to induce a preovulatory surge of LH as part of an ovulatory control system.

With this background, systems that are used currently in dairy cattle will be described. Objectives of this presentation are to examine potential impacts of reproductive technology to increase herd reproductive performance, and to emphasize that use of these new practices has to be founded on an understanding of the technology and its integration with good nutritional management.

Principles and Limitations of Implementing a Timed Insemination Program

Herd pregnancy rate is the product of heat detection rates and pregnancy rates to inseminations at detected heats (conception rate). Intensive research investigations have focused on trying to optimize these two biological components. One strategy to control heat detection rates is to control precisely the time of ovulation so that all cows can be inseminated at a fixed time, which is equivalent to a 100% heat detection rate. If our ability to precisely control ovulation time can be achieved in all cows and pregnancy rate to a timed insemination is normal, then a major advancement in reproductive management will have occurred. At the present time, there are only two drugs available to dairy producers for use in lactating dairy cows. Prostaglandin F2α (PGF2α) drugs (e.g., Lutalyse, Pharmacia Upjohn) can be used effectively to regress the corpus luteum (CL) but are ineffective on CL that are developing on days 1 to 5 of the estrous cycle. The other major drug is the Gonadotrophin Releasing Hormone (GnRH) like drugs (e.g., Cystorelin, Merial Co.) that release both LH and FSH from the pituitary of the cow. GnRH has the ability to ovulate a mature follicle to form a CL, and induce recruitment of a new follicle. Research at the University of Florida demonstrated that injection of GnRH recruits development of a new dominant follicle, which will induce the cow to express estrus when PGF2α is injected 7 days later (Macmillan and Thatcher, 1991). Investigators at the University of Wisconsin demonstrated that an additional treatment with GnRH after injection of PGF2α would induce a timed ovulation (Pursley et al., 1995). This procedure is known as the Ovsynch Program because it synchronizes ovulation and permits a timed insemination (Pursley et al., 1997). Pioneering studies at the University of Wisconsin and University of Florida demonstrated that in lactating dairy cows pregnancy rates are normal following a timed insemination (see review by Risco et al., 1999). However, the challenge is to further optimize this system based on an understanding of how the system works and recognizing the physiological constraints that limit performance of the system.

Figure 1. Plasma progesterone and follicle dynamics during anOvsynch/TAI program started on day 5 of the estrous cycle.

The Ovsynch protocol is idealized in Figure 1. In this example, concentrations of progesterone are monitored to document presence of a CL (the CL produces progesterone), and an idealized description of follicle development is presented. In this example, the cow is injected with GnRH (Monday, 5:00 PM) on day 5 of the estrous cycle. At this time, the cow has a dominant and healthy follicle that ovulates in response to the GnRH-induced release of LH; furthermore, the increase in FSH induced by the GnRH injection induces recruitment of a new pool of follicles in approximately 2 days (day 7) and one of the follicles is selected to become the dominant follicle (Moreira et al., 2000d). On day 12 of the cycle (7 days after the injection of GnRH), PG is injected (e.g., Monday, 5:00 PM) to regress both the original CL present at day 5 of the cycle and a newly formed CL that was induced by the injection of GnRH on day 5 of the cycle. The decrease in progesterone associated with regression of the CL accelerates growth of the newly recruited dominant follicle and a second injection of GnRH is made 2 days after the injection of PG (e.g., Wednesday, 5:00 PM). The second injection of GnRH induces ovulation 24 to 32 hours later (Pursley et al., 1995). Knowing that ovulation will occur at this time, the timed insemination is given at approximately 16 hours after the injection of GnRH (e.g., 9:00 AM on Thursday). This permits sufficient time for sperm to develop the capacity to fertilize the egg so that when it ovulates, a fertile population of sperm is present to carry out fertilization or initiate a pregnancy. This is an idealized scenario and the timing of injections is considered critical to the success of the program. If an interval of less then 7 days is used between GnRH and PG injection, the ability to effectively regress a newly developed CL is reduced. If the second injection of GnRH is delayed to longer than 48 hours or 2 days, then more cows are detected in heat prior to injection of GnRH, cows become asynchronized, and the timing of insemination is not correct. It is essential that producers not alter the protocol. One commonly asked question is, can cows be inseminated at the time of the GnRH injection or at 24 h after the injection of GnRH to make the insemination process more convenient? Pregnancy rates will be lowered at the 24 h insemination, and optimal insemination times appear to be between 12-18 hours after the GnRH injection (Pursley et al., 1998)

Success of the program is dependent on whether lactating dairy cows are cycling as well as stage of the estrous cycle at the time the Ovsynch protocol is initiated in cycling cows. Clearly, if cows in the group are not cycling then it is a given that pregnancy rates of the group will be somewhat less even though the Ovsynch protocol itself may induce some cows to begin to cycle and perhaps conceive. Figures 2 and 3 provide examples as to specific stages of the estrous cycle that lead to lower pregnancy rates when the Ovsynch protocol is initiated. In Figure 2, a cow initiates the program at day 15 of the estrous cycle when a normal second wave follicle is under development

INITIATION OF OVSYNCH/TAIAT LATE DIESTRUS

Figure 2. Plasma progesterone and follicle dynamics during anOvsynch/TAI program started on day 15 of the estrous cycle.

This follicle may or may not ovulate depending upon how mature it is. In many instances the second wave follicle is too small to ovulate and a new CL does not develop. At 2 to 4 days after the injection of GnRH, the cow spontaneously induces regression of the CL by releasing PG from the uterus. Thus, at the time of the PG injection given 7 days after the GnRH injection, the cow has already regressed the CL and may even be in heat (Moreira et al., 2000b). Such cows will be asynchronized in that they will ovulate prematurely; if we continue the protocol then insemination is made too late and the cow does not conceive. A second problematic stage of the cycle is in the early phases of the estrous cycle (e.g., day 2) as demonstrated in Figure 3. In this scenario, the cow already has been

INITIATION OF OVSYNCH/TAIAT METESTRUS

Figure 3. Plasma progesterone and follicle dynamics during anOvsynch/TAI program started on day 15 of the estrous cycle.

in heat, ovulated and is recruiting a new dominant follicle. This is a small follicle and the injection of GnRH on day 2 does not alter development of the dominant follicle and does not recruit a new dominant follicle (Moreira et al., 2000b). As a consequence, at the time of the second GnRH injection, the dominant follicle is considered aged and has expressed dominance for 5 days or longer. Follicles that have periods of dominance longer than 5 days (Austin et al., 1999) or cows that initiate the Ovsynch program in early stages of the estrous cycle are less fertile (Moreira et al., 2000b; Vasconcelos et al., 1999). Follicles may ovulate but oocytes are less fertile, or some cows may fail to ovulate their follicle in response to the second injection of GnRH. We can project what the success rate of the Ovsynch program will be in an idealized situation in which all cows are cycling and are at random stages of the estrous cycle when the program is initiated (Table 1.). Assuming a 20-day estrous cycle, we would expect 5% of the cows to be at each day of the estrous cycle. Thus, for a group of 100 cows, the percent of cows in early stages of the cycle (problematic), early diestrus, late diestrus (problematic) and proestrus are depicted in Table 1 with expected pregnancy rates at each stage for the reasons described above. An expected overall pregnancy rate of 36% is anticipated. However, it is

(Table 1. Herd distribution, expected pregnancy rates (PR), andpregnancies (Preg) in a 100 cyclic cow herd initiating theOvsynch/TAI protocol at random stages of the estrous cycle.)

Day of the cycle	Herd distribution	Expected PR	Preg in a 100 cow herd
1 to 4	20%	20%	4
5 to 12	40%	50%	20
13 to 17	25%	20%	5
18 to 20	15%	50%	7
Total	100%	—	36%

possible to manipulate the estrous cycle of cows such that they are in the ideal stage of the estrous cycle when the Ovsynch program is initiated. One idealized scenario is to inject all cows twice with PG at an interval of 14 days and to initiate the first GnRH injection of the Ovsynch program on day 12 after the second injection of PG. Such an idealized manipulation is demonstrated in Table 2. If all cows were cycling, we would

(Table 2. Herd distribution, expected pregnancy rates (PR), andpregnancies (Preg) in a 100 cyclic cow herd after pre-synchronization through two PG injections 14 days apart (the2ndPG is given 12 days prior to initiation of the Ovsynch protocol).)

Day of the cycle	Herd distribution	Expected PR	Preg in a 100 cow herd
5 to 10	90%	50%	45
13 to 17	5%	20%	1
18 to 20	5%	50%	2
Total	100%	—	48%

expect 90% of the cows to be in the ideal stage of the estrous cycle, between 5 to 10 days, when the Ovsynch program is initiated 12 days after the second injection of PG. With this scenario, an expected pregnancy rate to the Ovsynch program is 48%. Such a proposed treatment program prior to implementation of the Ovsynch program is called pre-synchronization with a standard protocol that is practiced in the industry. It is imperative that the producer and veterinarian have a thorough understanding of the principles of ovarian manipulation in order to understand how the system functions when they make herd management decisions as to how to implement the program.

Impact of Body Condition Score On Pregnancy Rates To The Ovsynch/TAI Program

There is the perception that pregnancy rates are lower in lactating dairy cows with poor body condition. Retrospective analyses of our field experiments indicate that as body condition score increases pregnancy rate increases to the Ovsynch/TAI program (Burke et al., 1996). We recently completed an experiment that examined pregnancy rates of the Ovsynch/TAI program in cows that had Body Condition Scores (BCS) of <2.5 versus >2.5 (Moreira et al., 2000d). Pregnancy rates at days 27 and 45 after insemination were 18.1% and 11.1% for cows with a low BCS (81 cows) versus higher rates of 33.8% and 25.6% for cows with BCS >2.5. The proportion of cows conceiving to the first synchronized service was lower for the cows in low body condition, and this was a temporary decrease since rates of cumulative pregnancies during the ensuing 120

(Figure 4. Cumulative pregnancy rates from the first TI until 120d postpartum.)

days postpartum were similar (Figure 4). This demonstrates the importance of optimizing fertility to the first service. Utilizing the differences in pregnancy rates in cows with body condition scores <2.5 versus >2.5, dynamic modeling was used to estimate net revenue per cow per year when considering what percentage of the herd had a low BCS of <2.5 (Figure 5). The difference in net revenue was $10.33 per cow per year as to whether 10% versus 30% of the herd had low body condition scores at the time the Ovsynch/TAI program was initiated (Moreira et al., 2000d). Thus, it is essential that producers try to nutritionally manage the dynamics of body condition postpartum to optimize fertility rates. Why does a low body condition score result in a lower pregnancy rate to the Ovsynch/TAI program? Is the rate of anestrus (non-cycling cows) responsible for the poor pregnancy rates or are pregnancy rates in cows cycling reduced due to defective eggs and/or the reproductive tract is unable to maintain a pregnancy? These are researchable questions that warrant further investigation and some further insight on this issue is described later in the paper.

Figure 5. Estimates of additional revenue using different scenarios based on the percentage of cows in the herd with low BCS (<2.5).

Optimization Of Reproductive Performance With Pre-Synchronization Implemented Prior To The Ovsynch/TAI Program and Use of Bovine Somatotropin

Of concern to dairy producers is whether Bovine Somatotropin (bST; Posilac, Monsanto, St. Louis, MO; 500 mg) treatment can be initiated in the ninth week of lactation and be continued without compromising reproductive performance. Our previous research findings (Moreira et al., 2000c) indicated that first service pregnancy rates to the Ovsynch/TAI protocol were increased when cows received bST treatment. Furthermore, the same experiment provided no evidence that bST treatment had any detrimental effect on subsequent services and reproductive performance at 120 or 305 days postpartum. Treatment with bST was initiated at day 63 postpartum concurrently with first injection of GnRH given as part of the Ovsynch/TAI program. An additional challenge is to document whether pregnancy rates to the Ovsynch/TAI program can be improved with prior implementation of a pre-synchronization program. A field trial was conducted with the objectives of: determining whether pre-synchronization of lactating cows prior to the initiation of the Ovsynch/TAI program would improve pregnancy rates; to verify prior results indicating that bST increased pregnancy rates to the Ovsynch/TAI program; to determine whether the possible beneficial effect of bST on pregnancy rates occurred prior to or after timed artificial insemination (Moreira et al., 2001). Development and implementation of the Ovsynch/TAI program has provided investigators a means to methodically test various factors that may improve pregnancy rates. Measuring the impact of any therapy or management system on pregnancy rates is a challenge because the experimental response is pregnancy rate in which a considerable number of cows are needed to detect potential differences, and the investigator has to cope with numerous management factors. At least with the Ovsynch/TAI program, the management errors associated with heat detection are eliminated and the precise timing of insemination can be controlled tightly.

Experimental design and treatment of lactating dairy cows:

The experimental design for pre-synchronization is depicted in Figure 6. A total of 543 cows were assigned randomly to the experiment in which half of the cows received the pre-synchronization program. The pre-synchronization program was

EXPERIMENTAL DESIGN

initiated on a weekly basis such that cows 34 to 40 days postpartum (37+ 3 days) received an injection of PGF2α (Lutalyse, Pharmacia-Upjohn Co.; 25 mg; i.m.) and this was followed 14 days later (51 +3 days) with a second PGF2α injection. In contrast, control cows (no pre-synchronization) did not receive the two injections of PGF2α. The rationale for the pre-synchronization program is described above and potential impact on pregnancy rates presented in Table 2. On day 63 + 3 days, the first injection of GnRH of the Ovsynch/TAI program was initiated, and this was 12 days after the second injection of PGF2α of the pre-synchronization program. The pre-synchronization program will place cows between days 5 to 10 of the cycle at the time of the GnRH injection depending upon what day they expressed estrus after the second injection of PGF2α (Figure 7). Days 5 to 10 of the cycle are considered by us as an optimal time to begin the Ovsynch/TAI program as discussed above. Following the protocols depicted in Figure 6, all cows will be time inseminated at day 73 + 3 days postpartum. The other factors tested in this experiment are the initiation of bST treatment at day 63 (time of the GnRH injection of the Ovsynch/TAI program), day 73 (time of artificial insemination as part of the Ovsynch/TAI program) or bST-control in which first injection of bST was not given until 147 days of lactation (well after first and second services). With this experimental design, the array of six treatment groups and number of cows in each group are shown in Figure 8. A series of blood samples were collected on days 51, 63, 70 72 and 79. Relative comparisons of progesterone concentrations in plasma allow us to determine if

PRE-SYNCHRONIZATION TREATMEN

Figure 7. Pre-synchronization effect on day of cycle at time of GnRHinjection.

cows are cycling (samples on days 51 and 63), stage of the cycle at the beginning of the Ovsynch/TAI program (samples on days 63 and 70), whether CL regression was successfully induced (samples on days 70 and 72) and whether cows had a synchronized ovulation (samples on days 72 and 79). All cows were examined by ultrasonography for pregnancy on day 32 after timed insemination and pregnant cows re-examined for pregnancy by rectal palpation on day 74 after timed insemination. This allowed us to characterize fetal losses between 32 and 74 days of pregnancy. All cows diagnosed open at day 32 after the first timed insemination were injected with GnRH and the Ovsynch/TAI program repeated with second insemination occurring at 115 days of lactation.

Impact of Anestrous Cows: With our ability to measure plasma progesterone in two plasma samples collected 12 days apart (on days 51 and 63 postpartum), it is possible to

EXPERIMENTAL DESIGNbST

Figure 8. Treatment groups that received either a pre-synchronization (+/-) and/or bSTat 63 or 73 days postpartum (number of cows)

identify exactly what cows are anestrus when the Ovsynch/TAI program is initiated. If cows had progesterone <1ng/ml in both samples they were considered to be anestrus. It was important for us to determine which cows are cycling since pre-synchronization treatments and potential effects of bST on pregnancy rates will not occur in cows that are not cycling. Furthermore, this assessment of anestrous status will allow us to document the frequency of this condition and its impact on reproductive performance of the herd.

For the assessment of anestrous status, 499 cows had blood samples collected on both days 51 and 63 postpartum. It is interesting that overall 23.4% of the cows were anestrus or had not started to cycle by 63 days postpartum (Table 3). Not surprising is the observation that the frequency of anestrus was greater for primiparous or first-calf heifers than multiparous cows (two lactations or more). The frequency of anestrus also was associated with body condition score as shown in Figure 9. The occurrence of anestrus decreased as body condition scores recorded at initiation of the Ovsynch/TAI improved. Therefore, body condition may be used to estimate the relative nutritional status of lactating dairy cows, and its impact on the frequency of anestrous cows at initiation of a reproductive management system.

Table 3. Percentage of cows classified asanestrusor cyclicaccording to plasma progesterone samples collected at 51DIM (second PGF2αinjection) and at 63 DIM (firstGnRHinjection of theOvsynch/TAI protocol) based on parity(cut off set at 1.0ng/ml).

Day of the cycle	Herd distribution	Expected PR	Preg in a 100 cow herd
5 to 10	90%	50%	45
13 to 17	5%	20%	1
18 to 20	5%	50%	2
Total	100%	—	48%

Figure 9. Relationship between body condition scores and frequency of anestrous cows at 63 days postpartum.

Body condition scores could only account for 7.8% of the variation in occurrence of anestrus. Thus, body condition score is not an absolute predictor of what cows are cycling. Some cows with body condition scores of 3.0 were anestrus. As anticipated, anestrous cows did not perform as well as cyclic cows in terms of pregnancy rates to the first-service Ovsynch/TAI protocol. Pregnancy rate at 74 days after insemination was only 22.4% for anestrous cows, which was lower than the 41.7% pregnancy rate at 74 days after insemination for cyclic cows. Since cows in anestrus do not respond to injections of PGF2α, pre-synchronization did not affect pregnancy rates of anestrous cows. Also, pregnancy rates of anestrous cows following first-service Ovsynch/TAI were not affected by administration of bST. Collectively, these results indicate that use of advanced technology to tightly control the reproductive cycle and stimulate embryonic development to increase pregnancy rates may not produce the expected results if there is a high frequency of anestrous cows at breeding. Therefore, postpartum management of lactating dairy cows is of extreme importance and will greatly affect reproductive performance. Efforts to maximize cow health, comfort, and nutritional status following parturition (e.g., enhance dry matter intake) will be reflected later in the lactation in terms of a higher incidence of cycling cows and improved reproductive performance.

Reproductive performance of Cyclic Cows:

Since anestrus had such a highly significant affect on pregnancy rates, reproductive performance was examined only in cyclic cows. First-service pregnancy rates to the Ovsynch/TAI protocol were affected by both bST pre-synchronization and bST treatments (Figure 10). Cows initiating bST treatment at 63 or at 73 days postpartum had increased pregnancy rates compared to controls among cows not pre-synchronized and also among cows pre-synchronized. The fact that a similar stimulation in pregnancy rates was observed in cows treated with bST at 63 (day of first GnRH injection) and at 73 days postpartum (day of timed insemination) indicates that bST is probably enhancing embryonic development and survival following insemination. Concentrations of bST are elevated throughout the 14-day period between injections such that bST injection at the time of the first GnRH injection will still elevate concentrations of bST 10 days later when the cow was inseminated. Since pregnancy rate was elevated when the first injection of bST was delayed to the time of insemination, the effects of bST appear to be targeted on the reproductive tract or on the embryo directly to enhance embryo survival.

Moreover, increased pregnancy rates were detected when cows were pre-synchronized (Figure 10; gray bars; 52.3%) compared to cows not pre-synchronized (white bars; 31.1%). An additional comparison is the effect of pre-synchronization in the two groups that did not receive bST in which pre-synchronized cows had a 42.6% pregnancy rate compared to 25.3% for the control group (Figure 10). This difference associated with pre-synchronization (17.3%) approximates the predicted differences (12.0%) that were estimated in Tables 1 and 2.

The reason for increased pregnancy rates to the Ovsynch/TAI protocol in cows pre-synchronized was related to the frequency of cows initiating the synchronization protocol at favorable stages of the estrous cycle. As indicated above, it was hypothesized that pregnancy rates to the Ovsynch/TAI protocol would be increased if cows received the first GnRH injection between days 5 to 10 of the cycle, and that pre-synchronization would synchronize approximately 90% of the cycling cows such that these cows would

Figure 10. First-service pregnancy rates to the Ovsynch/TAI protocol for cyclic cows (n = 375; LSM + SE).

be between days 5 and 10 of the cycle when the Ovsynch/TAI protocol was initiated. By collecting blood samples at the first injection of GnRH (at day 63) and again when PGF2α was injected (at day 73), we were able to indirectly identify cows that initiated the synchronization program at the early luteal phases of the estrous cycle (e.g., between days 5 to 10 of the cycle). Cows with high plasma progesterone (> 1.0 ng/ml) at both day 63 and day 73 (i.e., HIGH-HIGH cows) probably initiated the Ovsynch/TAI protocol at the optimal stage of the estrous cycle. Results from the frequency of cows classified as HIGH-HIGH indicated that approximately 87.4% of pre-synchronized cows were classified as HIGH-HIGH versus only 71.7% of cows not pre-synchronized were HIGH-HIGH cows (Figure 11). Therefore, we were successful in programming the cows to be in the optimal stage of the cycle to begin the Ovsynch/TAI program. As a consequence, pre-synchronization increased first-service synchronized pregnancy rates by enhancing the rate of synchronized ovulations and this increased the percentage of inseminated cows to respond to bST treatments. In a study by Cartmill et al., (2001) a single PG injection at 12 days prior to initiating the Ovsynch protocol increased synchronized pregnancy rate in multiparous cows. This was due to an increase in cows being in early diestrus at onset of the Ovsynch protocol.

Figure 11. Frequency of cyclic cows classified as HIGH-HIGH among treatment groups. More cows pre-synchronized were classified as HIGH-HIGH than cows not pre-synchronized.

Results from this experiment indicate for the second time that bST increased first-service pregnancy rates to the Ovsynch/TAI protocol. Such an observation impels us to review previous reports of decreased reproductive performance in cows receiving bST (Cole et al., 1992; Collier et al., 1997) and find explanations for such a discrepancy. It has been reported that use of bST may reduce the rate of estrous detection (Kirby et al., 1997) that may reduce reproductive performance of lactating cows. However, when estrous detection was eliminated though the use of the Ovsynch/TAI protocol, the inefficiency of heat detection possibly associated with bST may have been eliminated. We surely found no evidence that bST had any detrimental effect on reproductive performance in our studies. In contrast, pregnancy rates were increased at first service to timed inseminations as part of an Ovsynch/TAI program.

Since bST increased pregnancy rates to the timed insemination, several possible mechanisms may contribute to this effect and they appear to occur after timed insemination. Thus, a model for the possible effects of bST is depicted in Figure 12.

Figure 12. Model for the possible effects of bST on fertility of lactating cows.

The bST may be affecting final development of the ovulatory follicle after insemination (circle 1). Previous research has indicated that maturation of the oocyte in vitro is enhanced by both bST and IGF-I (Izadyar et al., 1996; Izadyar et al., 1997). Furthermore, bST also may be increasing plasma progesterone following insemination (circle 2) as demonstrated previously (Lucy et al., 1994). Increased plasma progesterone after insemination has been associated with greater pregnancy rates (Thatcher et al., 1994; Butler et al., 1996). However, in the present study we failed to see any difference in plasma progesterone concentrations measured at 6 days after the timed insemination. Studies in vitro indicated that embryonic development was enhanced by IGF-I (Palma et al., 1997), and embryonic development was accelerated in superovulated cows treated with bST (Moreira et al., 2000a). Thus, a direct effect of bST or an indirect effect of bST via IGF-I may be stimulating embryonic development and survival (circle 3). Also, treatment with bST may decrease the production of PGF2α by the endometrium at the time the embryo decreases secretion of PGF2α to maintain the CL (defined here as pregnancy recognition (circle 4; Badinga et al., 2000). This potential effect would increase the chances for embryo survival. All those are possible mechanisms that need to be further investigated to clarify the physiological processes that are influenced by bST treatment to increase pregnancy rates at first service to the Ovsynch/TAI protocol. Use of bST by dairy producers in coordination with the reproductive management system may enhance herd reproductive performance of lactating cows at first service and such a strategy provides the opportunity to further maximize the return on investment for the use of bST.

Potential uses of an Intravaginal Progesterone Releasing Device

Future availability of a device to release progesterone in dairy cattle will provide new and more efficient reproductive management options to the dairy industry beyond what can be achieved with the current uses of GnRH and PGF2α. Most strategies to control the estrous cycle employ an injection of PGF2α that regresses the corpus luteum (CL). Regression of the CL (luteolysis) is followed by the continued development of a preovulatory follicle that has been induced by a previous injection of GnRH as described above. Cows can be inseminated at detected estrus or receive a timed insemination preceding an ovulation induced by GnRH. However, PGF2α will not regress developing CL that are present on the ovary during the first 5 d of the estrous cycle (Lauderdale, 1972) or newly formed CL induced by a GnRH injection. Classically, one method to improve synchrony of estrus after a single injection of PGF2α is to treat cattle with a progestogen for 7 d before PGF2α (Macmillan and Peterson, 1993). Administration of the progestogen for 7 d before PGF2α ensures that CL will regress in response to PGF2α because all cattle will have a CL that has developed for at least 7 d. The progestogen will also delay estrus in cattle that naturally undergo CL regression during the progestogen treatment period before PGF2α injection (Roche et al., 1999). Furthermore, optimization of the Ovsynch/TAI program requires that cows start the program in early diestrus between days 5 to 12 of the estrous cycle. This was achieved with two injections of PGF2α given 14 days apart and beginning the Ovsynch/TAI program 12 days after the second injection of PGF2α, as described above. An alternative stategy would be to utilize a progesterone device that is inserted at the time of the first GnRH injection in the Ovsynch/TAI program. This would provide progesterone exposure to prevent early occurrences of estrus but not necessarily alter stage of follicular development at the beginning of such a program. A major limitation to the success of any estrus synchronization or ovulation control program is the presence of anestrous cattle or prepubertal heifers in the breeding herd. Progestogens offer an advantage in this regard because, in addition to improving estrus synchronization, progestogens will initiate estrus and ovulation in a percentage of prepubertal heifers and anestrous cows (Anderson et al., 1996; Fike et al., 1997; Imwalle et al., 1998).

Recently, a study was conducted to test the efficacy of an intravaginal insert containing progesterone and an injection of PGF2α for synchronizing estrus and shortening the interval to pregnancy in dairy heifers (Lucy et al., 2001). Multiple locations were used that employed the same protocol so that the efficacy could be evaluated in a variety of regions within the United States. Holstein dairy heifers (n = 260) that were 1 to 2 yr of age at the start of the trial were used. The heifers were at one of four locations (New York [n = 50], Illinois [n = 32], Missouri [n = 55], and Florida [n = 123]). Seven d before the start of the trial (d –7), heifers were bled and a second blood sample collected on d 0. Plasma concentrations of progesterone were used retrospectively to assign heifers to either prepubertal or cyclic groups. Heifers with progesterone concentrations at or below 1 ng/ml for both samples (d -7 and d 0) were declared as prepubertal heifers at the start of the experiment. Heifers with plasma progesterone concentrations above 1 ng/ml at either or both samples were designated as cyclic heifers. Control heifers were treated with a single i.m. injection of PGF2α (33.5 mg dinoprost tromethamine per 5 mL solution equivalent to 25 mg PGF2α; Lutalyse; Pharmacia and Upjohn Company, Kalamazoo, MI). The IPRI+PGF2α-treated heifers were administered an intravaginal progesterone-releasing insert containing 1.38 g progesterone (IPRI, Interag, Hamilton, NZ) for 7 d and injected i.m. with 25 mg PGF2α on d 6. The IPRI is a T-shaped insert and was placed into the vagina by using a lubricated applicator. The applicator collapses the wings for insertion into the vagina. Expulsion of the IPRI within the vagina causes relaxation of the wings and retention of the IPRI within the vagina by pressure on the vaginal wall. A thin nylon tail attached to the end of the IPRI is exteriorized through the vaginal opening and is used to remove the insert at the completion of the treatment period. The PGF2α injections were given on the same day for cattle assigned to the control group receiving PGF2α alone and the IPRI+PGF2α treatment. Estrous detection began on the morning of d 8 (2 d after PGF2α injection and 1 d after IPRI removal) and continued twice daily at 12 h intervals for 31 d. Cattle were observed morning and evening for at least 30 min at approximately 12-h intervals. Aids for estrous detection (tail paint, pressure sensitive patches, etc.) were used at the discretion of individual investigators for each region. Cattle were artificially inseminated approximately one half day after observed estrus with semen of known fertility. Semen from different bulls (if used) was blocked across treatments. Repeat services were given to those cattle returning to estrus after their first service. Pregnancy was diagnosed per rectum at 45 to 70 d after artificial insemination by using either ultrasonography or manual palpation. At the time of removal, the IPRI was not present in 5% of IPRI+PGF2α-treated heifers. The assumption is that the IPRI fell out of the vagina at some time during the treatment period. The cattle that lost IPRI were excluded from statistical analyses. Twelve dairy heifers located at the Florida site (12%; 12/123) were diagnosed as prepubertal at the time of IPRI insertion on d0.

There was an effect of treatment (P < 0.001) on the percentage of dairy heifers in estrus within the first 3 d of the breeding period (Table 4). Location was not significant. The majority of PGF2α-treated dairy heifers were in estrus on d 1 whereas the majority of IPRI+PGF2α-treated dairy heifers were in estrus on d 2 (Figure 13a). Across all locations, the percentages of dairy heifers in estrus during the first 3 d for PGF2α and IPRI+PGF2α groups were 57 (79/138) and 84% (103/122), respectively. Thus treatment with the IPRI markedly increased the synchrony of estrus during the 3 day target period. The effects of treatment and location were not significant for the percentage of dairy heifers detected in estrus during the entire 31-d breeding period (92%; 238/260). Thus the IPRI+PGF2α treatment accelerated the occurrence of estrus during the breeding period. Conception rate to inseminations during the first 3 d of the breeding period was not affected by treatment but tended to be affected by location (P < 0.10; Table 4). The conception rate for the first 3 d was 59% (106/180). Pregnancy rate during the first 3 d of the breeding period was not affected by treatment but was affected by location (P < 0.05). Across all locations, the percentage of dairy heifers pregnant within the first 3 d of the breeding period was 41% (106/258). In three of the four locations pregnancy rate was slightly higher for the IPRI+PGF2α group.

Conception rate and pregnancy rates to first inseminations during the entire 31-d breeding period were not affected by treatment but they were affected by location (P < 0.01). Across all locations, the first service conception rate was 54% (126/233) for the 31-day breeding period. The percentage of pregnant dairy heifers at the end of the 31-d breeding period was 58% (148/255). There was no effect of treatment on the survival

Table 4. The numbers of dairy heifers whose estruses were synchronized, conception rate, and pregnancy rate during the first 3 d of the breeding period.a

Item	New York	Illinois	Florida	Missouri	All Locations
Number Synchronized
PGF2a	16/30(53)	9/16(56)	34/63(54)	20/29(69)	79/138(57)
IPRI+PGF2a	17/20(85)	11/16(69)	52/60(87)	23/26(88)	103/122(84)
Conception rate c
PGF 2a	12/16(75)	4/9(44)	18/34(53)	17/19(89)	51/78)65)
IPRI+PGF 2a	11/17(65)	5/11(45)	26/51(51)	13/23(57)	55/102(54)
Pregnancy rate d
PGF 2a	12/30(40)	4/16(25)	18/63(29)	17/28(61)	51/137(37)
IPRI+PGF 2a	11/20(55)	5/16(31)	26/59(44)	13/26(50)	55/121(45)

aTreatments were PGF2α (single injection of 25 mg of PGF2α) or IPRI+PGF2α (1.38 g progesterone insert [IPRI] for 7 d with a single injection of 25 mg PGF2α on d 6). bnumber in estrus/number treated, %. cnumber pregnant/number inseminated, %.

curves for dairy heifers that were not pregnant during the 31-d breeding period (Figure 13B). However, there was a trend for the frequency of non-pregnant heifers to be lower for the IPRI+PGF2α group.

Of interest was that of the 12 heifers designated as anestrus (6 in PGF2α and 6 in IPRI+PGF2α groups) at the Florida location plus an additional 6 anestrous heifers treated with PGF2α at the Florida location, the IPRI+PGF2α group induced a greater percentage of synchronized heats (100%[6/6] > 25%[3/12]) and higher pregnancy rates for the 3 day (66%[4/6] > 16.6%[2/12]) and 31 day (66%[4/6] > 41.6%[5/12]) periods than anestrous heifers treated with PGF2α alone. This effect of progesterone on the ovary involves increased LH secretion that causes an increase in follicular development leading to ovulation (Anderson et al., 1996; Imwalle et al., 1998). The success of the IPRI + PGF treatment to induce a fertile estrus in prepubertal animals will depend upon how close the heifers are to the time of puberty. Puberty induction depends upon the heifers being in a transition period in which sensitivity to estradiol negative feedback is reduced, and progesterone priming appears to accelerate this transition such that a LH surge can be induced following withdrawl of the IPRI device. The effect of the IPRI+PGF2α treatment to induce estrus in non-cycling heifers confirmed the advantages of progestogen treatment for cattle that are not cyclic at the beginning of the breeding season (Patterson et al., 1989; Odde, 1990; Larson and Ball, 1992; Macmillan and Peterson, 1993).

Figure 13. The proportion of dairy heifers either not observed in estrus (A) or not pregnant (B) on each day during the 31-d breeding period (Survival analyses). Heifers were treated with PGF2α (single injection of 25 mg of PGF2a) or IPRI+PG.

The overall response to the IPRI+PGF2α treatment in a breeding program will depend on the proportion of cattle that are cyclic at the time the program is initiated, with the most desirable response occurring in potential breeding groups with greater percentages of cyclic cattle. Additional research is warranted to examine whether IPRI+PGF2α treatment can successfully induce cyclicity in postpartum anestrous lactating dairy cows.

Potentially Novel New Uses of the Intravaginal Progesterone Insert (IPRI)

Future availability of a IPRI device in dairy heifers and lactating dairy cows offers novel uses to potentially increase reproductive efficiency. In addition to synchronization of estrus and application to timed insemination programs, the IPRI may be reinserted at specific times after insemination to possibly increase embryo survival. Increased progesterone during the early period of CL development may accelerate embryo development. Likewise, insertion of an IPRI during the later luteal phases (e.g., day 7) may increase progesterone and enhance embryo survival. One of the biggest problems facing dairy producers is the long interval to second service following first service. Insertion of an IPRI device at 13 days after insemination for a 7 day period may aid in the re-synchronization of dairy cows that have not conceived. These applications will require extensive testing with dairy cows and may need to be combined with other drugs (e.g., GnRH and Estradiol Cypionate) to optimize their effectiveness. As mentioned above, the IPRI+PGF2α program appears to offer promise to treat both anestrus cows and pre-pubertal dairy heifers. An additional use of an IPRI is the treatment of cows with ovarian follicular cysts. Progesterone exposure contributes to turnover of the cyst and permits induction of a preovulatory LH surge upon withdrawal of the IPRI device.

Use of Estradiol Cypionate for Timed Insemination

One of the uses of exogenous estradiol as part of estrus synchronization systems is based on estradiol’s ability to induce a LH surge by stimulating hypothalamic secretion of GnRH. Estradiol also increases pituitary sensitivity to GnRH, apparently by increasing the number of GnRH receptors within the pituitary. Estradiol treatments during late diestrus and proestrus (low progesterone environment) will induce preovulatory surges of LH and FSH (Stumpf et al., 1991; Kinder et al., 1991). The preovulatory surge induced by an injection of estradiol at proestrus lasts for approximately 10 h (Short et al., 1979), which is similar to the spontaneous surge. Indeed these LH surges are of a greater duration than that induced by GnRH.

Estradiol cypionate (ECP), an esterified form of estradiol-17β, is approved for use in lactating dairy cows in the United States. We have conducted a series of experiments to evaluate whether Estradiol Cypionate could be used as part of a timed insemination program in dairy animals to replace the second GnRH injection of an Ovsynch/TAI program (Lopes et al., 2000; Pancarci et al., 2001).

Dairy Heifers: All experimental animals were synchronized with injection of GnRH (100 µg) on day 0, fed Melengestrol Acetate (MGA) on days 1-6 (0.5mg/d) and injected with PGF2α on day 7 (Lopes et al., 2000). Estradiol Cypionate (0.5 mg) was injected i.m. on day 8 in 14 dairy heifers and time of ovulation determined by ultrasound at 6 h intervals beginning at 48 h after Estradiol Cypionate injection. Ovulation occurred at 62 ± 2h after Estradiol Cypionate. A field trial was conducted to examine pregnancy rates to a Timed Insemination following injection of Estradiol Cypionate. Heifers (n=158) were assigned to control or Estradiol Cypionate timed insemination groups in August, 1999 during the period of seasonal heat stress in South Florida. Following synchronization (GnRH, MGA, PGF2α as described above), control heifers were inseminated at estrus, and Estradiol Cypionate treated heifers were injected with 0.5mg Estradiol Cypionate on d8 and timed inseminated 48 h later on day 10. Pregnancy rates did not differ between control (39.2%) and Estradiol Cypionate treated (39.2%) heifers. However a decrease in pregnancy rates was observed for the heifers that initiated the synchronization treatment at late diestrus and were timed inseminated. These two experiments indicated that Estradiol Cypionate (0.5 mg) was able to induce a synchronized ovulation with normal pregnancy rates in dairy heifers.

A second field trial was conducted in March, 2000 in South Florida prior to the heat stress season to test whether pregnancy rates to the Estradiol cypionate -TAI system could be increased by extending the MGA feeding until day 7 (day of PGF2α injection). All experimental heifers (n=160) were synchronized with GnRH (100 µg) on day 0, 25 mg of PGF2α on day 7, ovulation was induced by an injection of 0.5 mg of Estradiol cypionate on day 8, and insemination was performed at a fixed time of 48 h following Estradiol cypionate injection. Heifers were divided randomly into two treatment groups: MGA feeding from d 1 to d 6 (n=79) or from d 1 to d 7 (n=81). Blood samples were collected on day 0 and day 7 to characterize cyclicity and/or stage of the cycle of the heifers at initiation of treatment. Pregnancy diagnosis was performed by rectal palpation at 55 d after insemination. No differences in pregnancy rate were observed among the groups that received MGA from days 1 to d 6 (46.8%) or from days 1 to d 7 (48.1%). Furthermore, the decrease in pregnancy observed previously in the heifers that initiated the treatment in late diestrus also was observed in the present experiment (p<0.02), and was not avoided by extending MGA feeding by one day. This decrease in pregnancy rate could have resulted from either a premature ovulation, due to a failure of MGA to avoid a premature drop in progesterone, or from a possible reduced fertility of an aged follicle/oocyte that was induced to ovulate by the Estradiol cypionate injection. Estradiol cypionate can be used effectively as part of a TAI system, in dairy heifers, resulting in good pregnancy rates. All heifers received a fixed timed insemination with pregnancy rates approaching 50%. Further studies are required to address the reduction in pregnancy rates observed in late diestrus animals. Current studies are underway to examine the effects of Estradiol Cypionate as part of a timed insemination protocol in and results will be discussed.

Lactating Dairy Cows:

The use of ECP within the Ovsynch protocol may offer dairy producers an alternative, cost efficient reproductive management system if effective in lactating dairy cows. Several experiments were conducted in lactating dairy cows with the following objectives:1) to determine whether ECP can replace the second GnRH injection of a TAI protocol by evaluating pregnancy rate to first service in lactating dairy cows and 2) to characterize the time of induced estrus and ovulation after injection of ECP in lactating dairy cows during proestrus (Pancarci et al., 2001).

Experiment 1 evaluated pregnancy rates when estradiol cypionate (ECP) was used to induce ovulation as part of a timed artificial insemination (TAI) protocol in comparison to Ovsynch for lactating dairy cows in Florida (n=371) and Texas (n=321). Cows were pre-synchronized with two injections of PG given 14 d apart with TAI protocols beginning 14 d after the second injection of PG. The TAI protocols consisted of an injection of GnRH followed by PG 7d later. Cows were injected either with GnRH (Treatment I, Ovsynch) at 48 h after PG and inseminated 16-24 h later or with ECP (1 mg, i.m.) at 24 h after PG, (Treatment II; Heatsynch) and inseminated 48 h later. In Florida, pregnancy rates after TAI were 37.1 + 5.8% for Ovsynch compared to 35.1 + 5.0% for Heatsynch (Table 5). Pregnancy rate to first insemination was higher for primiparous (47.1%) cows than multiparous (25.0%) cows (P<0.01). Primiparous cows by design were not inseminated until 100 days postpartum versus 78 days postpartum for multiparous cows. There was a tendency for a parity by treatment interaction (P<0.10) in which pregnancy rates did not differ between Ovsynch and Heatsynch in primiparous cows, but pregnancy rate tended to be lower for the Heatsynch group in multiparous cows (Table 5). It is important to recognize that any difference in parity of the Florida study is confounded with differences in days postpartum for TAI (primiparous, 100 days versus multiparous, 78 days).

Table 5. Pregnancy rates of primiparous and multiparous cows at 46 + 3 d after insemination (LSM + SE; Florida site).

Parity	Ovsynch(n=179)	Heatsynch Group	Overall(n=371)
Primiparous(n=201)	43.5+-6.9(n=100)	50.7+-6.3(n=101)	47.1+-5.6
Multiparous(n=170)	30.6+-7.2(n=79)	19.4+-6.5(n=91)	25.0+-5.7
Overall(n=371)	37.1+-5.8	35.1+-5.0	39.6+-2.5 a,b

a,bTreatment x Parity Interaction, P<.10; Parity, P<0.01 In Texas, pregnancy rates were 28.2 + 3.6 % for Ovsynch and 29.0 + 3.5 % for Heatsynch. Overall pregnancy rates did not differ between Ovsynch and Heatsynch treatments. No difference in pregnancy rate was detected between primiparous (40.6%; n=105) and multiparous (34.7%; n=216) cows that were inseminated at the same days postpartum (i.e., 73 days). An interaction between treatment and whether cows were in estrus at TAI (P<0.05) was detected in which cows in estrus had a higher pregnancy rate in the Heatsynch group; whereas, pregnancy rates were comparable in estrus and non-estrus cows of the Ovsynch group (Figure 14). Occurrence of estrus on the day of TAI was greater in the Heatsynch group (65.2%>17.8%, P<0.01). Since no difference was detected in pregnancy rate due to parity, it is possible that a delay in TAI for first calf heifers may have improved fertility in the Florida study.

Figure 14. Pregnancy rates at 37 days after TAI (LSM + SE) in lactating dairy cows that were in estrus or not in estrus at the time of insemination following Ovsynch and Heatsynch treatments.

In Experiment 2, estrus (monitored by the Heat Watch system) and ovulation times were determined in lactating dairy cows submitted to the Heatsynch protocol (Pancarci et al., 2001). Frequencies of detected estrus and ovulation after ECP were 75.7% (28/37) and 86.5% (32/37), respectively. Mean intervals to ovulation were 55.4 + 2.7 h (n=32) after ECP and 27.5 + 1.1 h (n=27) after onset of estrus. Estrus occurred at 29.0 + 1.8 h (n=28) after ECP. It is recommended that any cow detected in estrus by 24 h after ECP injection be inseminated at 24 h, and all remaining cows be inseminated at 48 h, since 75% (n=24/32) of the ovulations occurred between >48 h to <72 h after ECP. Synchronization of ovulation and fertility results indicated that ECP could be utilized to induce ovulation for a timed insemination. Based on synchronization of ovulation and pregnancy rates, ECP can be utilized to induce ovulation in place of GnRH for a timed insemination. Timing of ECP injection to induce ovulation for a timed insemination differs compared to the use of GnRH. Greater uterine tone, ease of insemination and occurrence of estrus improve acceptance by the inseminator. On the other hand, in facilities with poor footing the reduced estrous expression following GnRH may be preferred. This timed insemination system with the use of ECP is referred to as “Heat Synch”.

Future investigations are needed to evaluate whether Estradiol Cypionate can be used to: induce follicular turnover in the beginning of a Timed Insemination protocol and be used as part of a re-synchronization program for second service in cows that did not conceive to the first service.

Improved Embryo Survival

Early embryonic losses have been documented in numerous studies in cattle. Factors such as dairy versus beef cattle, fertile versus repeat breeder cows, insemination to spontaneous versus synchronized estrus, season of year relative to heat stress conditions, and other factors such as parity, nutrition and disease contribute to the time of embryonic losses. It is clear that infertile cows (e.g., repeat breeder cows) or groups of animals in which the proportion of infertile cows is high experience appreciable embryonic losses (~30%) by day 7 of pregnancy. In more fertile groups of animals, embryonic losses (~40%) occur gradually between days 8 to 17 of gestation (Thatcher et al., 1994). When the transfer of morphologically normal embryos eliminated fertilization failure and early embryonic losses, 24.4% of recipient cows terminated their pregnancies between day 17 and 24 (Markette et al., 1985). The rate of late embryonic mortality after day 27 in a fertile group of dairy heifers was estimated to be 10.6% (Thatcher et al., 1994) and this agrees with an estimate of 10.5% in lactating dairy cows that were pregnant at 28 d and lost a pregnancy by day 42 (Vasconcelos et al., 1997). However, it is interesting that these later stages of embryonic losses appear greater in recent estimates with high producing dairy cows as depicted in the above experiment involving body condition. Collectively, these reports indicate differential timing of embryonic losses and a failure of different mechanisms to sustain embryo development.

The need for close synchrony between embryonic and uterine development of which P4 is a primary regulator is important. Optimal fertility was obtained if donor and recipients were in estrus the same day. A critical question is whether variation in embryo survival in cattle is related to the post-ovulatory rise in P4. Subfertile cows have a slower rate of P4rise during the 6-day post-ovulatory period. Cows bearing normal embryos have greater plasma P4 concentration on day 3 post-estrus. Lower concentrations of plasma progesterone at approximately day 12 after insemination have been reported for cattle in which early pregnancy fails. Thus a low rate of progesterone rise following ovulation and lower luteal phase concentrations of progesterone are associated with reduced embryo survival (see review, Thatcher et al., 1994). Pritchard et al., (1994) reported that concentrations of estradiol during days 14 to 17 after breeding in beef cattle might be associated with embryonic losses. Cows were divided into three groups according to concentrations of estradiol, the lower quarter, middle half and upper quarter, which averaged 1.6, 2.1 and 3.1 pg/ml of estradiol, respectively, during the 4-day sampling period. Conception rate to first service by artificial insemination was related linearly to the groupings in which conception rate decreased as concentration of estradiol in plasma increased. Mean conception rates were 77, 60 and 42 %, respectively. This raises the interesting possibility that ovarian follicle development may be associated with embryo survival. Follicular development is sustained on the ovary bearing the CL in hysterectomized cows with prolonged CL function (Thatcher et al., 1991). In contrast,

follicular development was attenuated on the CL bearing ovary of heifers during the same period of pregnancy. Thus the conceptus and/or ipsilateral gravid uterine horn, and not the CL decrease intraovarian follicular development in a local manner. Such an effect would be supportive of an antiluteolytic mechanism for maintenance of pregnancy. Ahmad et al. (1997) reported that perhaps dynamics of follicular wave patterns might differ between cows that conceive or remain open after insemination. Pregnant animals tended to switch from having two follicular waves during the estrous cycle before breeding to having three waves during the equivalent period immediately after breeding. In contrast, 8 of 9 nonpregnant beef animals had two follicular waves in both the estrous cycle before insemination and the equivalent period after breeding. Fewer animals conceived among those that had two (70%) rather than three (96%; P < 0.05) waves of follicular development during the period after insemination.

Diaz et al., (1998) has developed a strategy to increase the luteal phase concentrations of progesterone and induce a three wave follicular cycle by injecting hCG at day 5 after estrus. All heifers injected with hCG formed an accessory CL, and luteal phase concentrations of progesterone were elevated by day 9. All hCG treated heifers had three follicular waves in which the dominant follicle of the third follicular wave did not reach 9 to 10 mm in size until approximately day 20. Dominant follicles do not develop appreciable expression of LH receptors in granulosa cells until greater than 9 mm in size (Bao and Garverick, 1998). Thus the potential to secrete estradiol is limited until follicles exceed 10 mm in size. With this rationale, the potential estrogenic follicle for hCG treated heifers would not occur until d 20 versus d 14 in control heifers with a two wave follicular cycle (Diaz et al., 1998). Therefore hCG treatment would decrease the estrogenic environment during the period of pregnancy recognition. Injection of hCG on day 7 has increased conception rates in lactating dairy cows (Sianangama and Rajamahendran, 1992) with a modest increase in plasma progesterone concentrations. To date, direct progesterone supplementation during the early luteal and mid luteal phases of the estrous cycle have not given consistent beneficial effects on pregnancy rates. It is likely that systems of progesterone delivery are needed that increases both the rate of progesterone rise and absolute luteal phase concentrations of progesterone after insemination. Further efforts to influence CL differentiation and subsequent function is a fruitful area of investigation

Use of hCG to increase pregnancy rate

cows were housed separately. Cows were fed a total mixed ration twice daily ad libitum to meet or exceed the requirements for a lactating cow that weighs 650 kg and produces 45 kg of milk with 3.5% fat. The nutrient composition of the diet was 1.78 Mcal of NEL, and 18.6% crude protein, 6.4% ruminally undegradable protein, 6.1% fat, and 31.3% neutral detergent fiber as percentage of the total diet DM.

The study was divided into two periods: period 1 of May 14 to September 16, 1999 when daily maximum temperatures ranged from 22 to 38 °C (warm period), and period 2 from October 5, 1999 to March 02, 2000, when daily maximum temperatures ranged from 9 to 29 °C (cool period). One hundred and eighty-two cows were enrolled during the warm period and 224 cows during the cool period. Once a week, a group of 30 to 60 cows between 40 and 145 days in milk had their estrous cycle synchronized with an i.m. injection of 100 µg of GnRH (Gonadorelin; Factrel, Fort Dodge Animal Health Inc., Fort Dodge, IA) followed 7 days later by an i.m. injection of 25 mg of PGF2α (Lutalyse, Pharmacia Upjohn Company, Kalamazoo, MI). Estrus was detected once daily, from 2 to 5 days after the PGF2α injection, both visually and by tail chalking (Macmillan et al., 1988). Cows in estrus were assigned to initiate the experiment based on lactation number and days in milk. Seventy two % of the treated cows were detected in estrus. Therefore, about 20 to 40 cows were enrolled in the study every week. All cows were artificially inseminated once daily, in the morning, by the same technician throughout the experimental period. Semen from five different proven sires was used and a similar number of doses of semen of each sire were utilized in each treatment group. Cows found open at day 28 received an injection of 25 mg of PGF2α and were artificially inseminated as detected in estrus within 5 days of the prostaglandin treatment. For analyses of the second service conception rates, only cows that were inseminated in the subsequent estrus at 18 to 24 days after the initial AI and those that came in estrus after the PGF2α injection at day 28 were evaluated.

On day 5 after AI, cows received an i.m. injection in the neck area of either 3,300 IU (hCG group) of hCG (Chorulon®, INTERVET, Inc., Millsboro, DE) or 3 ml of saline solution (control group). One blood sample was collected from all cows between days 11 and 16 after insemination and subsequently analyzed for concentrations of plasma progesterone. At the same time of blood sampling, the ovaries of all cows were scanned by ultrasound to evaluate presence, location (ovary), size (area and volume), and number of CL. Diagnosis of pregnancy was performed by ultrasonography on day 28±1 after AI. During ultrasonography, a cow was determined pregnant when an embryonic vesicle with a viable embryo (presence of heart beat) was detected. At the same time, ovaries of pregnant cows were scanned to determine the presence and location of the ovulatory and accessory CL. Pregnant cows at day 28 were reconfirmed by rectal palpation on day 45±1, and again on day 90±2 after AI. At the time of AI and on day’s 28±1, 45±1, and 90±2 after insemination, cows were body condition scored by the same person. To evaluate changes in BCS, cows were divided into three groups in which cows lost, had no change, or gained BCS. Milk production from the test date closest to the day of AI (day of AI ± 10 day) was divided into four quartiles or into two groups (above or below the mean, 43.0 kg/day) and utilized to test the effects of milk yield on conception rates and other responses. The range in milk production for the first, second, third and fourth quartiles were 20.4 to 36.3 kg/day, 36.3 to 41.7 kg/day, 41.7 to 49.9 kg/day, and 49.9 to 69.0 kg/day, respectively.

Administration of 3,300 IU of hCG on day 5 after AI successfully induced the formation of one or more accessory CL in 175 of the 203 treated cows. More cows in the hCG-treated group had multiples CL (86.2% vs 23.2%; P < 0.001). The increase in number of CL for cows treated with hCG compared with control cows was greater during the cool than during the warm period (P < 0.02). Number of CL on day 14 was not influenced by BCS at breeding. However, the change in BCS between AI and day 28 after breeding affected CL number (P < 0.01), with cows losing BCS having the highest mean number of CL, followed by those that did not change or gained BCS in the same period.

Plasma progesterone concentrations during the mid luteal phase were increased in the hCG-treated cows compared with the control cows (P < 0.0001). Differences in progesterone concentrations between hCG and control cows were +10.6 ng/ml for primiparous cows and +7.5 ng/ml for multiparous cows (P< 0.02). Cows with more observable CL during ultrasonography had higher progesterone concentrations (P < 0.0001), and an interaction between CL number and treatment on plasma progesterone was observed (P < 0.0001). Within the hCG group, cows with more than 1 CL had higher plasma progesterone than those with only 1 CL, and it was increased by 9.8 ng/ml. Such an effect of accessory CL on progesterone concentrations was not observed for cows in the control group, in which progesterone levels were similar.

At day 28 post AI, CR was increased from 38.7% for control cows to 45.8% for the hCG-treated cows (P < 0.01; Table 6). Similar CR was observed during the warm and the cool periods, but a tendency for an interaction between treatment and period was detected (P < 0.09). Control and hCG-treated cows had similar CR during the warm period, but hCG increased CR compared with control during the cool period (47.8 vs 34.2%). Cows with 0, 1 or more than 1 CL on d 14 had CR of 0, 31.7, and 53.6%, respectively (P < 0.0001). Plasma progesterone in pregnant cows was 3 ng/ml higher than in cows that were open at day 28 (18.2 vs 15.2 ng/ml; P < 0.001). Similar effects of progesterone were observed for CR at 45 and 90 days post AI.

Table 6. Effect of treatment on CR at days 28, 45 and 90 post AI.

Treatment1
Conception rate,%	Control	hCG	P<
Day 28	38.7	45.8	0.01
Day 45	36.3	40.4	0.005
Day 90	31.9	38.4	0.008

When milk production was divided into quartiles, cows with higher production tended to have lower CR (P < 0.09). Conception rates decreased from 53.4% to 41.4, 38.8, and 35.9% for the first, second, third and fourth quartiles of milk yield. Cows with moderate BCS at the time of AI had higher CR than cows with low BCS (P < 0.001). Conception rates on day 28 were 34.4 and 48.7% for cows with low and moderate BCS, and these effects were similar for control and hCG-treated cows. Similar to the results observed for BCS at the day of AI, changes in body score from AI to day 28 were also associated with changes in CR. Cows that gained BCS from AI to day 28 had higher pregnancy than those that lost or maintained BCS (47.0% vs 42.7% vs 37.4%; P < 0.03). Interestingly, an interaction between treatment and BCS change was determined for pregnancy at day 28. Cows that lost BCS when treated with hCG had a CR of 57.1% compared with only 24.2% for those in the control group (P < 0.05). When pregnancy diagnosis was considered at day 45 and day 90 after AI, the effects of treatment, number of CL, plasma progesterone concentration, BCS at the day of AI, and BCS change were similar to those observed for CR at day 28 after insemination.

For the second service CR analyses, only 196 of the 234 cows that were re-inseminated or found open and recycled at day 28 were included in the analyses. Although treated cows had a CR of 34.1% compared with 30.6% for the control group, hCG treatment on day 5 of the previous estrous cycle had no effect on subsequent fertility (P < 0.56). The only tendency observed for CR at the second service was an increase in pregnancy for cows that were gaining BCS from the beginning of the study to day 28 compared with those that lost or did not change body condition (40.8% vs 25.7% vs 27.1%; P< 0.14). Concentrations of progesterone during mid luteal phase of the previous cycle were similar for cows that conceived or were open after the second AI and averaged 14.8 ng/ml.

Pregnancy loss was determined from days 28 to 45, 45 to 90 and the overall loss from 28 to 90 days. Only cows that were initially found pregnant and subsequently found open were included in these analyses. For pregnancy losses between 28 and 45 days after insemination, the data from 172 cows were utilized, 79 in the control group and 93 in the hCG-treated group. Cows in the hCG-treated group had a slightly higher pregnancy loss during this period, but no statistical difference was detected (11.8 vs 6.3; P < 0.21). Number of CL on day 14 influenced pregnancy loss from 28 to 45 days. Increasing the number of CL from 1 to more than 1 decreased pregnancy loss from 13.2% to 7.6% (P < 0.007). In addition to CL number, an interaction between treatment and period was observed. Control cows lost more pregnancies during the warm period (7.3 vs 5.3%) compared with hCG cows that lost more during the cool period (10.3 vs 13.0%) (P < 0.008). Data from 156 (74 control and 82 hCG) cows were utilized to analyze pregnancy losses between days 45 and 90 postinsemination. Percentages of cows that lost pregnancy in the hCG and control groups were 4.9 and 13.5%, respectively (P<.10) Overall pregnancy loss from day 28 to day 90 was similar for hCG and control cows, and averaged 17.4%.

This study supports the concept that increased progesterone during the luteal phase increases embryo survival. However, this effect was not evident in the heat stress period where early embryo losses probably precluded any subsequent increases in embryo survival.

Implications for Dairy Producers

A vast array of options has been recently developed for the reproductive management of lactating dairy cows. Such management systems have been fine-tuned to result in maximum pregnancy rates and increase the overall reproductive efficiency of lactating dairy herds. As it can be observed in the experiments described above, pregnancy rates as great as 50% to a single service were achieved. It is important to emphasize, that as reproductive systems become more efficient and incorporate several levels for the control of reproductive processes, a thorough understanding of the technology is needed.

It is important for producers to realize that such reproductive management systems cannot solve all reproductive problems per se. For instance, incidence of cows in anestrus greatly reduces the reproductive efficiency of dairy herds and such a problem may not be solved by synchronization systems. Providing optimal nutritional management, maximizing cow comfort, and maintaining a good herd health program are all pre-requisites for the success of any reproductive program.

To date, management of dairy cows has been driven by the necessity to maximize milk production with a high overall success. With the advent of new technologies to precisely manipulate reproductive function in lactating dairy cows, dairy producers are presented with a new opportunity. Coordination of management strategies to maximize both milk production and reproductive performance may optimize the economical return of dairy herds, and allow for the industry to take complete advantage of the genetic potential to improve milk production through artificial insemination. Further research is necessary to fine tune management systems that are both practical and able to fulfill this objective. Producers can look forward to new strategies to reduce anestrus, synchronize return services and enhance embryo survival.

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Source: Texas A&M University
Author: Thatcher