Introduction

Considerable research has focused on developing technologies to synchronize and efficiently detect estrus (Larson and Ball, 1992; Sterry et al., 2007), as lactating dairy cows have poor reproductive performance due to low fertility and low rates of estrus detection. Recently, a protocol has been developed (ovsynch) that synchronize the time of ovulation precisely using GnRH and PGF2α (Pursley et al., 1995). Ovsynch is based on the initiation of a new follicular wave with GnRH before inducing luteolysis by PGF2α. The emerging dominant follicle is forced to ovulate by a second treatment with GnRH allowing for timed artificial insemination (TAI). Ovsynch protocol utilizes 3 treatments; GnRH administered at a random stage of the estrus cycle, PGF2α given 7 days later, and a 2 ^nd GnRH treatment 2 days after PGF2α injection. In lactating dairy cows, this protocol synchronized follicular development, regression of the corpus luteum, and time of ovulation allowing effective TAI without the need for detection of estrus (Burke et al., 1996; Schmitt et al., 1996; Pursley et al., 1997). Combinations of PGF2α, steroids (E2 and P4) and GnRH are used to regulate the life span of the corpus luteum, control follicular wave emergence and ovulation, and synchronize estrus in cattle (Larson and Ball, 1992; Thatcher, 2001). Prostaglandins (PGF2α) or its analogues induce luteal regression, but interval to estrus is highly variable in randomly cycling cows (Lauderdale et al., 1974). Short-term pretreatment with P4 or multiple injections of PGF2α (at 11-14 day interval) reduced the proportion of cows that are in met estrus and increases the percentage of cows in estrus (Britt, 1979; Xu et al., 1997; Bicalho et al., 2007). A more precise expression of estrus (after PGF2α) can be achieved by synchronizing the follicular wave that gives rise to the preovulatory follicle. Estrogens and GnRH induce atresia or ovulation of dominant follicles and synchronize the emergence of a new follicular wave (Bo et al., 1994; Burke et al., 1999). Substituting oestradiol cypionate (ECP) for the second GnRH injection synchronizes ovulation and estrus and yields similar pregnancy rates to the original ovsynch program (Lopes et al., 2000; Pancarci et al., 2002). Oestradiol cypionate is the only form of estradiol registered for use in lactating dairy cows in the USA. Kojima et al. (2000) tested a GnRH-PGF2α protocol combined with P4 pretreatment in postpartum beef cows.

The objectives of the present study are, a) to evaluate an estrus synchronization protocol for dairy cows based on the 7-11 system, as the interval elapsed between the GnRH and 2 ^nd PGF2α injection was prolonged to 8 days (longer than the traditional 7 days interval, b) to improve the synchrony of estrus and ovulation, after placement of an ECP at the end of treatment.

Material and Methods

Animals

Treatments design

Treatment 1 (PP)

Treatment 2 (PGP)

Treatment 3 (PGPE-0)

Treatment 4 (PGPE-1)

Estrus detection and artificial insemination

Assay of plasma progesterone (P4) and estradiol (E2)

Pregnancy check

Cows were checked for pregnancy by ultrasound scanning at 40 and confirmed by rectal palpation at 45 days after artificial insemination. The submission, conception and pregnancy rates were measured in the four groups of treated cows.

Submission rate=Number of cows inseminated within 4 days after 2 ^nd PGF2α injection/total number of cows.

Conception rate=Number of pregnant cows /number of inseminated cows.

Pregnancy rate=Number of pregnant cows /total number of treated cows.

Figure 1

Figure 1: Time line for treatment administration (PGF=2 ml Estrumate; GnRH=100 ug Cystorelin; ECP=1 mg Estradiol cypionate). Estrus detection and artificial insemination for cows treated with PP, PGP, PGPE-0 and PGPE-1 protocols. Blood plasma was collected for progesterone and estradiol analysis.

Statistical analysis

Results

A greater percentage of GnRH-treated cows (PGP) ovulated after 1 ^st PGF2α injection than the non treated (PP) cows (64.3% vs 50%; P<0.05; Table 1). The improved ovulation percentage was associated with a large follicle that tended (P<0.10) to be larger in size at the time of 2 ^nd PGF2α injection for PGP vs PP treated cows. However, mature follicles were determined by 2 days after 2 ^nd PGF2α injection. Although treatment with GnRH was associated with an increased ovulation percentage after 1 ^st PGF2α injection, the percentage of cows that ovulated after 2 ^nd PGF2α injection was 71.4% versus 50%; for GnRH-treated cows than non treated ones, respectively (Table 1). The interval to estrus (64±7 h) and the interval to ovulation (97±9 h) were also similar for both treatments.

Cows treated with PGP had higher peak preovulatory concentrations of estradiol in plasma compared to PP treated cows (3.2±0.48 vs 2.6±0.45 pg/ml) following the 2 ^nd PGF2α injection (Fig. 2).

After the 2 ^nd PGF2α injection, ovulation was defined as low P4 (<0.5 ng/ml) on day 13 and high P4 (≥0.5 ng/ml) on day 24. Cows not ovulated may had high P4 on day 13 and 24, low P4 on day 13 and 24, or high P4 on day 13 and low P4 on day 24. The development of accessory corpus luteum in the presence of a functional corpora lutea could not be determined because ovulation was based mainly on P4 concentration.

At the time of 2 ^nd PGF2α injection, the size of mature follicle was similar for all treatments after the ultrasound scanning. However, for cows treated with ECP (PGPE-0 and PGPE-1), mature follicles also determined within 3 days after 2 ^nd PGF2α injection (P<0.05). Addition of ECP to the PGP treatment increased the percentage of cows ovulating (50% and 71.4% for PP and PGP vs 75.0% and 87.5% for PGPE-0 and PGPE-1; P<0.05; Table 1). Thus, the percentage of ovulations for PGPE-0 and PGPE-1 treated cows tended to be higher compared to the other treated cows. Interval to oestrus and interval to ovulation were similar for all treatments (55±5 and 91±7 h, respectively). For those cows that ovulated, the actual days of ovulation after 2 ^nd PGF2α injection were 3, 4 and 4 days for PGP; 3, 3, 4, 4, 4 and 5 days for PGPE-0 and 3, 4, 4, 4, 4 and 4 days for PGPE-1 treatments.

Treatment with ECP also increased plasma estradiol concentration after 2 ^nd PGF2α injection (Fig. 3; P<0.05). Plasma estradiol concentrations were the greatest at 1 and 2 days after 2 ^nd PGF2α injection for PGPE-0 and PGPE-1 treated cows, respectively. In each case, the peak level of estradiol occurred one day after ECP injection. However, cows treated with ECP (PGPE-0 and PGPE-1) had a higher peak preovulatory concentrations of estradiol in plasma (6.3±0.43 and 6.99±0.63 pg/ml; P<0.01) following 2 ^nd PGF2α injection.

Thirty one of sixty cows (51.6%) were classified as anoestrus at the start of the trial (plasma P4 <0.5 ng/ml on days –8, 0 and 3). The percentage of cows that developed a corpus luteum (hereafter referred to as ovulation) after 1 ^st PGF2α injection was greater when cows received GnRH (75% and 81.2%; for PGPE-0 and PGPE-1 versus 50% and 64.3% for PP and PGP; P<0.001). A higher percentage of ECP treated cows were observed in standing oestrus and ovulated after 2 ^nd PGF2α injection and; 75% and 87.5%; P<0.05 for PGPE-0 and PGPE-1 vs 50% and71.4% for PP and PGP treated cows, respectively.

Plasma estradiol concentrations were similar across treatments at the time of 2 ^nd PGF2α injection. However, 2 days after 2 ^nd PGF2α, plasma estradiol concentrations were greater in cows that received ECP compared to PP or PGP (P<0.001; Fig. 2). The percentage of cows that ovulated (subsequently developed a corpus luteum) after 2 ^nd PGF2α injection was the greatest with ECP groups than the others (P<0.05).

Submission rates (number of inseminated cows/totalX100), the conception and pregnancy rates was observed to be higher with the ECP treated cows than the other treatments (Table 2).

Figure 2

Table 1: Summary of ovarian response to PGF2α, GnRH and estradiol cypionate injection for cows treated with four synchronization protocols.

Figure 3

Figure 2: Plasma estradiol concentrations (Least square means, SEM) after the 2 PGF2α injection for cows treated with PP (n=14), PGP (n=14), PGPE-0 (n=16) and PGPE-1 (n=16). There was a treatment by day interaction (P

Figure 4

Table 2: Submission, conception and pregnancy rates for cows treated with four synchronization protocols.

Discussion

An estrus synchronization protocol (termed PGP) similar to 7-11 synch (Kojima et al., 2000; Kanitz et al., 2006) but without Melongesterol acetate was developed. The protocol consists of a PGF2α injection followed 3 days later by a GnRH injection and another PGF2α injection 8 days after the GnRH. The protocol is similar to the original PGF2α protocol consisting of 2 injections of PGF2α 11 days a part (Lucy et al., 1986; Sterry et al., 2007; Bicalho et al., 2007), but includes an injection of GnRH 3 days after the first PGF2α injection. The GnRH provides two-fold advantages. First, it forces a timely ovulation of the dominant follicle after 1 ^st PGF2α injection (Thatcher and Chenault, 1976). The timed ovulation prevents late ovulations that would potentially result in an immature and unresponsive corpus luteum at the time of 2 ^nd PGF2α injection. Second, the GnRH forces a percentage of anoestrus cows to ovulate (Britt et al., 1974) and induces cyclicity in cows that would otherwise not respond to PGF2α. The advantage of PGP was examined in the present experiments. In each case, the number of cows that ovulated after 1 ^st PGF2α injection was increased. For example, the percentage of cows that ovulated after 1 ^st PGF2α injection developed when GnRH was given on day 3 (PGP and PGPE-1 compared to PP).

In the present study, the cows treated with PGP had mature follicles that were larger at the time of 2 ^nd PGF2α injection. However, 2 days later, follicles were similar in size because the mature follicles of PP-treated cows continued to grow, whereas those of PGP-treated cows remained the same size. Treatment with GnRH appears to produce more mature follicles at the time of 2 ^nd PGF2α injection; supported by the fact that both PGP and PGPE-1-treated cows were in estrus before PP-treated cows. Luteal and follicular maturity may be an advantage of the PGP protocol over traditional GnRH- PGF2α protocols. The extra day between the GnRH and PGF2α injection (8 days for PGP versus 7 days for traditional protocols) may place more cows in diestrum (being responsive to PGF2α), and more mature follicle may lead to a more functional CL upon ovulation (Vasconcelos et al., 2001; Kanitz et al., 2006; Sterry et al., 2007).

Addition of ECP increased the number of cows that ovulated after 2 ^nd PGF2α injection (Table 1) and improved the percentage of cows in standing estrus. Both responses were associated with increased plasma estradiol concentrations after 2 ^nd PGF2α injection. Although plasma estradiol concentrations were elevated, they were within physiological ranges and there were no incidences of false estrus (behavioral estrus without ovulation). Addition of oestradiol to PGP protocol also had the unexpected effect of increasing follicular growth. The suppressive effect of estrogens on follicular development is well documented (Bo et al., 1994; Burke et al., 2000; Martinez et al., 2002) but stimulatory effects on preovulatory follicular growth have not been reported in cattle (Burke et al., 2000). It appears that exogenous estradiol stimulated some aspects of dominant follicular growth but the exact mechanism is not known.

One major goal for any new estrus synchronization protocol should be the implementation of timed AI. The interval to ovulation and interval to estrus were examined in this study. The results were promising, as a higher percentage of GnRH-treated cows ovulated after 1 ^st PGF2α injection (64.3% vs 50%; P<0.05). The GnRH-treated cows tended to have a larger mature follicle present at 2 ^nd PGF2α injection. The percentage of cows that ovulated after 2 ^nd PGF2α injection was 71.4% vs 50% for GnRH-treated cows than none treated ones, respectively. Cows treated with PGP had higher peak preovulatory concentrations of estradiol in plasma compared to PP treated cows (3.2±0.48 vs 2.6±0.45 pg/ml; following 2 ^nd PGF2α injection. Cows treated with ECP (PGPE-0 and PGPE-1) had a significant higher peak preovulatory concentrations of estradiol (6.3±0.43 and 6.99±0.63 pg/ml); and a significant higher percentage ovulation (75.0% and 87.5%) than the other treated groups (PP or PGP). The PGP protocols are similar to the other GnRH-PGF2α protocols because GnRH and PGF2α are given in a series. The primary difference is that PGF2α is given before 1 ^st GnRH injection. The first injection of PGF2α causes corpus luteum regression so that most cows are on day 8 of the estrus cycle when 2 ^nd PGF2α dose is injected. In late luteal phase, cows treated with GnRH and PGF2α at a 7 day interval tend to exhibit estrus around the time of PGF2α injection; i.e. before the time when most cows are in estrus, 2-3 days after PGF2α (Vasconcelos et al., 1999; Dejarnette et al., 2001). The present study did not compare PGP to GnRH and PGF2α given at a 7 day interval but with one day more (8 days). Nevertheless, estrus was not observed before 2 ^nd PGF2α injection in PGP cows. The possibility that relative to GnRH and PGF2α-alone, PGP increases the percentage of cows in estrus 2-3 days after the last PGF2α injection should be tested in larger experiment.

GnRH is an obvious substitution for ECP in the PGPE-1 protocol. The GnRH would be given 48 hours after 2 ^nd PGF2α injection but the timing of other injections would be similar (PGF2α day 0, GnRH day 3, PGF2α day 11, GnRH day 13; PGPG protocol). In a previous study, the PGPG protocol was tested in 45 postpartum dairy cows and 93% of the cows ovulated on day 14 (Sterry et al., 2007). Thus, the PGPG protocol may be an alternative to PGPE-1 that is highly suited for timed AI.

In conclusion, we can conclude that PGP protocol increased the number of ovulated cows after 1 ^st PGF2α injection and produced more mature follicles at the time of 2 ^nd PGF2α injection. Adding ECP to PGP (especially PGPE-1) enhanced the expression of estrus and increased ovulation percentage. Thus, a combination of PGP and ECP is potentially a new method to routinely synchronize estrus and ovulation in dairy cows.