Timed artificial insemination : current challenges and recent advances in reproductive efficiency in beef and dairy herds in Brazil

Beef and dairy productivity depends directly on the reproductive efficiency and genetic gain of the herd, which can be related to the appropriate use of biotechnologies, such as timed artificial insemination (TAI). When considering variations in synchronizations protocols, longer or shorter periods of progesterone (P4) device treatment could provide benefits to fertility. However, our studies evidenced that protocols with six (J-synch), seven, eight and nine days of P4 device treatment had similar pregnancy per AI (P/AI). In cyclic cows, the early prostaglandin (PGF) administration, moving from the day of P4 device removal to two days earlier, which results in four handlings of cows, or the administration of one extra dose of PGF at the onset of the protocol and a single PGF on the day of P4 device removal (three handlings) are both efficient to induce early luteolysis, reducing serum P4 concentrations and, therefore, stimulating LH pulsatility, which improves growth of the dominant follicle and results greater P/AI when compared with protocols with the administration of PGF only on the day of P4 device removal. Resynchronization is another valuable tool to reduce the interval between AI. Traditional Resynch is initiated at pregnancy diagnosis (28 to 32 days after TAI) and the interval between AI is around 40 days; Resynch 22 and Resynch 14 respectively initiates 22 and 14 days after the previous AI in all cows (unknown status of pregnancy) and reduces the interval between AI to 32 and 24 days. The novelty about Resynch 14 is the need to use of Doppler ultrasonography for pregnancy diagnosis [evaluation of corpus luteum (CL) vascularization]. Similar P/AI after Resynch 22 and 14 were found in Nelore cows. In dairy cattle, reproductive management is carried out throughout the year, thus, it is important to adapt the reproductive management to few established days of the week. Therefore, traditional Resynch and Resynch 25 were set to start 32 and 25 days after previous TAI, respectively. The hastening of reproductive age of Nelore heifers aims to reduce age at first calving and increase productivity. Factors such as age, weight, body condition score (BCS), uterine score (USC), average daily weight gain (ADG), withers height/depth of rib relationship (dRIB) and subcutaneous fat thickness (SCFT) were associated with an increase in the success of gestational establishment at TAI and can be used to select the heifers that are more suitable for reproduction. These technologies can contribute to improve the national production of kilograms of meat and liters of milk per hectare, and consequently improve livestock profitability.


Introduction
Brazil has a distinguished position in the global beef industry.In addition to being one of the leaders in the beef export market, it is prominent in the scientific development and commercial application of applied reproductive biotechnologies.The correct use of biotechnologies in farms plays a critical role on productivity.Among the most used reproductive biotechnologies, timed artificial insemination (TAI)which eliminates the need for estrous detectiondeserves to be highlighted for facilitating management and by improving reproductive efficiency and genetic gain of the herds.Data comprised in our laboratory in 2016 showed that in 2015, TAI moved approximately R$567 million (~U$175 million) in Brazil, with an estimate of 3,500 veterinarians directly involved with this activity.Timed AI is currently implemented on 8.2 million beef cows, therefore generating an increase of 8% on calves' production, which represents approximately 656 thousand more calves per year or an additional income of R$820 million/year (~U$253 million) compared with natural service breeding.Time AI also hastens parturition and adds genetic gain to the herds, generating an average gain of 20 kg on the weaning weight of calves, which represents 3.3 million weaned calves with extra 20 kg or, extra R$400 million (~U$123 million).Also, from weaning to slaughtering, TAI calves gain an additional 15 kg of carcass, generating extra R$482.2 million (~U$149 million).Thus, TAI aggregates to the bovine beef chain around R$1.7 billion (more than half billion US $) per year (Baruselli, 2016).
As for dairy herds, TAI also has impact and adds around extra R$900 million (~U$278 million) per year by reducing the calving interval and using genetically superior bulls.It is estimated that TAI reduces one month of calving interval, increasing by 10% the annual milk production, what in Brazil represents extra 690 million liters of milk or R$759 million (~U$234 million) of additional income per year.Additionally, the use of bulls with superior genetics adds around 300 liters of milk per lactation of the daughter, resulting in an extra income of R$113.9 million (~U$35 million; Baruselli, 2016).Thus, it is estimated that the impact of TAI on dairy and beef chain together achieves around R$2.6 billion (~U$800 million) per year of extra income.
In 2016, TAI reached the mark of 11,034,119 procedures, which represents a growth of 5.1% in relation to the previous year (Fig. 1; non-published data from VRA-USP-Brazil; 2017), according to a carried out survey based on the number of protocols sold for TAI (information provided by companies in the sector) and the number of commercialized semen straws (Associação Brasileira de Inseminação Artificial -ASBIA, 2017).Currently, TAI procedures correspond to 85% of the insemination performed in Brazil (Fig. 1).Thus, it is evident that TAI holds a relevant place in the AI market.The strong progress made in recent years is indicative that the technology has been consolidated in the market, as it has positive results for livestock and qualified professionals for its execution.In spite of the proved benefits of TAI for reproductive efficiency, only 10 to 12% of females in reproductive age are in fact inseminated in Brazil (Baruselli, 2016).Thus, new strategies for expanding the AI application and optimizing the results should be incorporated to maximize its use and improve even more its economic impact.
Figure 1.Time evolution of artificial insemination (AI) in Brazil.The numbers of AI done after estrus detection and following treatments for timed AI (TAI) are estimated based on the number of protocols sold for TAI (information provided by companies in the sector) and the number of commercialized semen straws (ASBIA, 2017).Data was organized by Departamento de Reprodução Animal-FMVZ-USP, São Paulo, Brazil, 2017.

Recent conquests for improving the TAI efficiency in the field
In this topic, the evolution and main perspectives of TAI programs used in beef and dairy cattle in Brazil will be discussed.Variations of current synchronization protocols, such as duration of the progesterone (P4) device and early prostaglandin F2α (PGF) treatment, were investigated to optimize animal handling and the efficiency of TAI protocols.Additionally, the evolution of programs to resynchronize ovulation, allowing subsequent management of TAI during the breeding season may reduce the calving interval and accelerate the genetic gain.Finally, the reduction of the heifers' age as they enter TAI programs is another great tool to maximize the calve production of genetic superior females earlier, hastening the age at parturition and accelerating the genetic advance.

The evolution and flexibility of TAI protocols
Over the past years, TAI protocols for beef and dairy cattle have gone through several modifications aiming to improve pregnancy outcomes.The length of treatment with P4 or progestin devices (summarized in Table 1) and early administration of PGF (from the day of device removal to two days before device removal; summarized in Table 2) were the main alterations evaluated.Briefly, in most experiments, the basic protocol used as control was the insertion of a P4 or progestin-releasing device plus the administration IM of estradiol benzoate (EB) at random days of the estrous cycle defined as day 0, device removal and intramuscular administration of PGF, estradiol cypionate (EC) and equine chorionic gonadotropin (eCG) on day 8 and TAI 48 h later.

Year
The modifications of the duration of P4 or progestin device treatment in cows are based in the following evidences.The increased duration of device treatment, from 8 to 9 days, could provide more growth time to the dominant follicle, guaranteeing the ovulation of follicles with larger diameter with greater ovulation rates (Mantovani et al., 2005(Mantovani et al., , 2010;;Sá Filho et al., 2010) and, consequently generating larger corpus luteum (CL) with greater capacity of P4 synthesis (Mantovani et al., 2005).On the other hand, excessively long periods of follicular dominance may disrupt oocyte quality, resulting in reduced fertility (Cerri et al., 2009;Mihm et al., 1999;Roche et al., 1999;Lonergan, 2011).Another hypothesis is that the reduction of the period of P4 exposition (to six or seven days) may avoid adverse effects of follicular growth at the end of the protocol, extending proestrus and increasing estradiol concentration during this period (Bó et al., 2016).Briefly, none of these modifications tested and summarized in Table 1 improved pregnancy per artificial insemination (P/AI) following TAI.Protocols with six (J-synch), 7, 8, or 9 days of P4 device treatment were similarly efficient (Table 1).Similarly, previous studies have shown that the age of the ovulatory follicle did not influence P/AI in heifers and suckled beef cows (Abreu et al., 2014a, b).The absence of differences between protocols using P4/E2/eCG with various periods of P4 device treatment (6 to 9 days) may indicate that the new dominant follicle has a window of size and proestrus duration to ovulate an oocyte that is competent and suitable for fertilization.This would allow a flexible management for TAI protocols.
In countries that use GnRH and PGF based protocols, the beneficial effect of changing the duration of traditional Ovsynch protocol has been associated with greater circulating estradiol concentrations by prolonging the proestrus prior to ovulation and greater progesterone concentrations in the ensuing luteal phase, especially in the cows that do not ovulate after the first GnRH (Bridges et al., 2014).Furthermore, high estradiol concentrations in the proestrus period have been associated to a more appropriate uterine environment and smaller incidence of embryo loss (Jinks et al., 2013).
On the other hand, in the P4/E2/eCG-based protocol routinely used in Brazil, the lack of differences when using different periods for P4 device treatment may be due to diverse reasons: 1) the high efficiency in the synchronization of a new follicular wave at the beginning of the protocol; 2) the treatment with eCG to stimulate dominant follicle growth and to increase circulating P4 concentrations in the subsequent luteal phase; and 3) the treatment with estrogen that increase circulating estradiol concentrations prior to a synchronized ovulation.
The other proposal of protocol modification was related to earliness administration of PGF, from the day of device removal to two days earlier, aiming to induce early luteolysis and reduce serum P4 in cyclic cows.This would stimulate luteinizing hormone (LH) pulsatility and, consequently, improve the growth of the dominant follicle (Mantovani et al., 2005(Mantovani et al., , 2010)).In this context, greater P/AI was achieved when cycling Nelore cows (detection of CL at the onset of the protocol) received an early administration of PGF [(two days previous to device removal; day 7; 50.3% (86/171)]) compared with those receiving PGF on the day of device removal [day 9; 36.1% (56/155); P < 0.05].Such difference was not observed in cows without a CL at the beginning of the treatment for TAI (Meneghetti et al., 2009).Increased P/AI was also observed with early PGF administration (from day 9 to day 7) for nonlactating Nelore cows (Peres et al., 2009).In dairy cows, the early administration of PGF from day 8 to day 7 also improved fertility to TAI and embryo transfer (Pereira et al., 2013).
Despite improving P/AI, the early administration of PGF demands an extra day of animal handling, which is undesirable, especially in beef farms.An alternative strategy to reduce P4 serum concentration during TAI protocol of cows with CL avoiding the extra handling is the inclusion of an extra dose of PGF at the onset of the protocol, keeping the second dose on the day of device removal (Carvalho et al., 2008).In this context, several studies showed similar P/AI when cows were treated with a single PGF administration on day 7 (four animal handlings) or PGF on day 0 and again on the day of device removal (three animal handlings) for Nelore cows (Carvalhaes et al., 2016;Mingoti et al., 2016), Girolando heifers (Mendanha et al., 2012) and crossbred Nelore-Aberdeen Angus heifers (Colli et al., 2016), as summarized in Table 2. Thus, the inclusion of an extra dose of PGF at the onset of the protocol allows improving fertility by reducing P4 serum concentration during TAI protocols, without the need of an extra day of handling.

Resynchronization of ovulation (Resynch programs)
In order to achieve maximum reproductive efficiency, aggressive strategies to concentrate pregnancies early in the breeding season should be taken.For that, three steps should be considered: AI of all cows at the beginning of the breeding season, early identification of nonpregnant cows, and reinsemination of nonpregnant cows as early as possible.The most commonly management adopted to get nonpregnant cows pregnant soon after the first AI is the introduction of clean-up bulls for the remainder of the breeding season.However, modern alternatives known as Resynchronization Programs are a potential tool to reduce time for subsequent inseminations.
The term "resynchronization" or "Resynch" refers to the treatment for synchronization of follicular wave emergence and ovulation of a female that was previously subjected to TAI.The aim of the technique is to allow the subsequent AI of cows that have already gone through one or more AI without establishing pregnancy, eliminating the need for estrus detection, maximizing the use of selected bulls and improving reproductive efficiency associated with genetic gain.Consequently, it allows the reduction of the interval between AI and hastens postpartum conception, reducing the calving interval and accelerating herds' genetic evolution.
At first, the Resynch program (so called traditional Resynch) was initiated at pregnancy diagnosis around 28 to 32 days after TAI ( Stevenson et al., 2003;Marques et al., 2012Marques et al., , 2015)).Cows diagnosed as nonpregnant immediately initiated a new protocol for TAI.This Resynch management is flexible (starts at date chosen for pregnancy diagnosis) and treatments are only performed in nonpregnant cows.However, the interval between AI is around 40 days and is still considered too long by some technicians compared with bull exposure, when mating occurs around 21 days after TAI.Although bull mating allows the reduction of the interval between two consecutive services, the service rate depends on the estrus return (around 50%; Sá Filho et al., 2013).The evolution of Resynch programs allowed the reduction of the interval between AI to compact the period of breeding season and the interval between two subsequent parturitions, with the benefit of guaranteeing 100% service rate.To reach this goal, the treatments should start earlier than pregnancy diagnosis and therefore should be done in all cows.The Resynch 22 was developed to initiate 22 days after the previous AI, eight days before pregnancy diagnosis (Sá Filho et al., 2014).At that time, cows diagnosed as pregnant are excluded from the following treatments and nonpregnant cows continue the synchronization treatment.The advantage of Resynch 22 is to reduce the interval between AI to 32 days, however, the first treatment (P4 device and estradiol) should be done in all cows and pregnancy diagnosis must be performed in a fixed schedule, differently from the traditional Resynch.Although most of the farms that use the traditional Resynch have a prescheduled date for pregnancy diagnosis in order to start the resynch protocol as soon as possible, this date is not mandatory (anytime the pregnancy diagnosis is done, it is possible to start a new protocol in open cows).On the other hand, for Resynch 22 or 14, the date of pregnancy diagnosis is mandatory because the protocol was already started eight days before.
More recently, a new approach of early Resynch was proposed using the technology of color Doppler ultrasonography to perform an earlier nonpregnancy diagnosis by analyzing the vascular patterns of the CL (and not the presence of an embryonic vesicle in the uterus as usually done; Siqueira et al., 2013;Pugliesi et al., 2017).This management is called Resynch 14, because the treatment starts 14 days after the previous TAI, followed by pregnancy diagnosis eight days later (22 days after TAI) using Doppler ultrasonography (Vieira et al., 2014).Again, this Resynch protocol demands the initial treatment in all animals (unknown pregnancy status) and a mandatory fixed schedule for pregnancy diagnosis.Additionally, it demands specific ultrasound equipment and a welltrained technician to perform pregnancy diagnosis by the evaluation of CL vascularization grade.However, it allows an impacting reduction in the interval between AI to 24 days, which it close to what is achieved with an ideal 21 days service rate (Fig. 2).
Regarding the early diagnosis of nonpregnancy in cows using the color Doppler ultrasonography, high accuracy and close to 100% sensitivity were observed using only the CL vascularization patters as an indicative of luteolysis to identify nonpregnant dairy cows (Siqueira et al., 2013).As for beef cattle, 100% sensibility and 91% accuracy was reported when the association of CL vascularization and size were evaluated to diagnose early pregnancy (Pugliesi et al., 2014).Thus, color Doppler ultrasonography is considered an accurate tool to diagnose early pregnancy, because there is a low possibility of a wrong diagnostic of a pregnant cow as nonpregnant (close to 0% of false negatives).The occurrence of pregnancies diagnosed in a subsequent B mode ultrasonography exam as nonpregnancies may be related to long estrous cycles, pregnancy loss, or a lack of synchronization to the first TAI protocol, and not necessarily is due to a wrong pregnancy diagnosis.In these cases, the nonpregnant cows may start a subsequent synchronization of follicular wave emergence and TAI.
Some peculiarities are inherent to each Resynch protocol, specially related to the initial treatment.For the traditional Resynch and Resynch 22 the treatment starts 30 or 22 days after TAI, respectively, and it consists on the insertion of a P4 intravaginal device plus the administration of 2 mg estradiol benzoate (Pessoa et al., 2015).The dose of EB used in Resynch 22 was determined based on a study using 1,426 cows (768 B. taurus and 728 B. indicus; Pessoa et al., 2015).Pregnancy to the first TAI and pregnancy loss between 30 and 62 days following AI was similar between cows receiving 1 mg (44.0 and 3.8%) or 2 mg EB (44.0 and 5.5%) on D22.However, pregnancy to Resynch and cumulative pregnancy were greater (P < 0.01) in cows treated with 2 mg EB (47.3 and 68.2%) than with 1 mg EB (36.1 and 62.8%).This difference may be related to more effective follicle wave synchronization with 2 mg EB than 1 mg EB in cows (Caccia and Bó, 1998).However, for Resynch 14 the initial treatment starts 14 days after TAI and the administration of estradiol is replaced with the administration of 100 mg P4 IM.This is recommended based on previous studies showing that the use of EB 13 to 14 days after the previous AI induces luteolysis in some of the cows, reducing the previous AI conception rates (Cutaia et al., 2002;Vieira et al., 2014).This fact is not observed when estradiol is administered 22 days following AI for the Resynch 22 program, as reported previously ( Sá Filho et al., 2014;Pessoa et al., 2015).
These results are in agreement with previous data showing a reduced ability of the CL to produce P4 when females are treated with EB during mid-diestrus (Munro and Moore, 1985;El-Zarkouny and Stevenson, 2004).This might be related to the induction of PGF release driven by the increase on estradiol concentration (Thatcher et al., 1986;Araújo et al., 2009).Alternatively to estradiol, P4 can be employed to promote the atresia of the dominant follicle and the emergence of a new wave.In this context, Rezende et al. (2016) demonstrated the growth of a new follicular wave 3.0 ± 0.7 after treatment with 100 mg P4 IM in Nelore cows.Thus, synchronization of follicular wave emergence in Resynch 14 programs must be performed replacing EB with P4 on the first day of treatment.
P/AI after Resynch 22 and Resynch 14 were recently compared in 244 postpartum Nelore cows (Penteado et al., 2016).For that, cows subjected to the first TAI were allocated into one of the two Resynch programs, Resynch 22 (ECC = 3.0; n = 126) or Resynch 14 (ECC = 3.0; n = 118).Resynch 22 cows were treated with a P4 device and 2 mg EB IM 22 days after the previous AI (day 22).On day 30, the device was removed and pregnancy was diagnosed based on the presence or absence of an embryonic vesicle in the uterus (conventional ultrasonography).Nonpregnant cows had the P4 device removed and received 0.530 mg sodium cloprostenol (PGF), 1 mg estradiol cypionate and 300 IU of eCG IM, followed by TAI 48 h later on day 32.Resynch 14 cows were treated with a P4 device plus IM administration of 100 mg P4 (Afisterone®, HertapeCalier) 14 days after the previous AI (day 14).On day 22, pregnancy diagnosis was done by the assessment of CL vascularization using Collor Doppler ultrasonography.Cows with absence or low CL vascularization were considered open and proceeded the treatment (device removal, PGF, estradiol cypionate and eCG IM), and were TAI 48 h later on day 24.Cows with moderate or strong CL vascularity were considered pregnant and had the device removed without further treatments.Similar P/AI were observed for Resynch 22 and Resynch 14 cows following the first AI (48 vs. 53%; P = 0.57) and resynchronization (56 vs. 51%; P = 0.37), respectively.The cumulative pregnancy after 32 and 24 days of breeding season did not differ (P = 0.77) for Resynch 22 (77%; 97/126) and Resynch 14 cows (75%; 89/118), respectively.Besides keeping similar P/AI after subsequent TAI and reducing the interval between AI to 24 days, Resynch 14 considerably improved 21 days service rate from 66 to 87.5% in relation to Resynch 22 (Fig. 3).The benefits of using Resynch programs are leading to the adoption of management exclusively with TAI, eliminating the need for clean-up bull in several farms.The use of three consecutive TAI using Resynch 22 (3 TAI) had similar pregnancy rates than those achieved with bull exposure after two TAI using Resynch 22 (2 TAI + bull) and greater pregnancy rate than one TAI followed by bull exposure (1 TAI + bull; Crepaldi et al., 2017).In this study, it was possible to achieve 87.4% of cumulative pregnancy rate at the end of a 64-day breeding season after three TAI using Resynch 22 (Fig. 4).5).
Pregnancy diagnosis was based on CL area and vascularization.Heifers with an area of CL ≥2 cm² and/or ≥25% CL blood flow were diagnosed as pregnant.False positive heifers were those diagnosed as pregnant at Doppler evaluation 22 days after AI and then as nonpregnant at pregnancy confirmation 30 days after AI.The false positive rate was 14.8% (47/317) and the P/AI of these heifers for the second time inseminated in D48 was 40.4% (19/47), increasing 2% in P/AI of day 48 (3rd TAI + false positive, 35.6%, 78/219).
These studies demonstrate that modern Resynch programs seems to be feasible and efficient on reducing the period of breeding season with similar cumulative pregnancy outcomes as obtained with bull exposure in traditional 90-day breeding season.It also brings the advantage of improving the number of pregnant animals by AI, accelerating the farms' genetic gain.Additionally, it concentrates calving births in the favorable calving season, consequently improving the weaning weight and accelerating the use of young females in the subsequent breeding season.
For dairy cattle, the Resynch protocols follow the same basis as for beef cattle.However, differently from beef, dairy herds reproductive management is accomplished all over the year, usually lacking a delimitated breeding season, and animals are managed several times a day.Thus, it becomes crucial to adapt the reproductive management so they do not interfere with the other daily management employed in a dairy farm.A good strategy is to concentrate the reproductive-related activities into established days of the week.In this basis, the traditional Resynch and Resynch 25 were set to start 32 and 25 days after previous AI, respectively (Fig. 6).This standardization of reproductive activities in specific weekdays enables the establishment of a well-planned routine in dairy farms, as shown in Figure 6.
In order to calculate reproductive efficiency of dairy cows in a Resynch 25 program, we considered that the first service occurs 58 days after parturition (average), 30% P/AI until third service, 20% P/AI between the fourth and sixth services (sixth service was established as the animals' last service) and 15% of pregnancy loss between 30 and 60 days of pregnancy.In this simulation, the herd achieves 82% of pregnant cows at 246 days in milk, 110.4 days interval between parturition and conception and 12.9 months of calving interval, which can be considered good reproductive efficiency.Therefore, studies were conducted in order to verify the applicability and viability of Resynch 25 in dairy properties in Brazil.
In one study, the efficiency of the association between gonadotropin-releasing hormone (GnRH) or EB and P4 at the onset of Resynch 25 to synchronize the new wave of follicular growth in Holstein cows without previous pregnancy diagnosis was evaluated (Vasconcellos et al., 2014).The authors verified that both associations (GnRH+P4 or EB+P4) were effective to synchronize the new wave of follicular growth and ovulation.Given this information, a second study was conducted to verify the possible influence of the administration of 2 mg EB at the onset of Resynch 25 on the establishment and maintenance of the previous pregnancy in Holstein cows (Vieira et al., 2015) were observed in cows resynchronized with EB, GnRH or a combination of both.The CL vascularization was also accessed by Doppler ultrasonography every 48 h, between days 25 and 33 in 42 lactating cows used in the previous experiment [EB: n = 15; GnRH: n = 12; EB+GnRH: n = 15].It was found that the CL vascularization rate remained similar between the three groups during the experimental period (day 25: 76.1%, day 27: 79.1%, day 29: 77.5%, day 31: 76.3% and day 33: 77.1%).Thus, the resynch treatment using EB, GnRH or a combination of both after TAI results in similar P/AI after 1st TAI and after resynchronization.Also the administration of EB in pregnant lactating cows 25 days after AI does not induce pregnancy loss and does not compromise the CL vascularization.
Therefore, Resynch 25 can be an alternative for reproductive management of dairy properties.This program allows systematization and planning of the annual reproduction calendar of the farm, concentrating conceptions at the beginning of lactation.However, it is important to mention that other factors, such as nutrition and sanitary management, as well as organization and availability of qualified personal, may significantly influence the results of TAI programs with resynchronization in dairy herds.

Hasten of reproductive age of Nelore heifers
Many factors may influence hasten or delay in the sexual maturity of heifers, such as age, weight after weaning and development of the reproductive tract.Knowing the real impact of these factors on the reproductive efficiency would be determinant for the development of future strategies that would accelerate even more the onset of reproduction of Nelore heifers in Brazil.Thus, the need to establish indicators of body development that determine improvements on the reproductive efficiency of yearling zebu heifers is evident.The main objective is to reduce age at first calving and increase productivity in Brazils' beef herd.
Recently, Freitas (2015) evaluated the body development and functioning of the reproductive tract in relation to gestational success in 650 14-month-old (13.9 ± 0.03 months) Nelore heifers that underwent TAI protocols.The heifers were kept on a pasture-based system.They were evaluated during 10 days before the onset of TAI protocol (day -10 to day 0).The evaluated characteristics were age (months), weight (kg, weight scale for squeeze chute), body condition score (BCS, 1 to 5 scale), withers height (hWIT, cm) and withers height/depth of rib relationship (dRIB, %), reproductive tract score (RTS, 1 to 5 scale), cyclicity (presence of a CL) and subcutaneous fat thickness (SCFT, mm).All heifers were synchronized for TAI (Norgestomet year implant + 2 mg EB -8d -PGF + 300 IU eCG + 0,6 mg estradiol cypionate -AI 48 hs).The cyclicity (presence of CL at day -10 and/or day 0) and P/AI 30 days after TAI were determined by ultrasonography.Briefly, hWIT and RTS were not associated with an increase in the success of gestational establishment.However, heifers that were older, heavier, had greater BCS, dRIB and SCFT had greater P/AI (Table 3).It was found that older females showed greater success in pregnancy (> 13.8 months = 43.0 vs. ≤ 13.8 months = 27.2%;P = 0.04).No association was observed between hWIT and P/AI (> 119.0 cm = 34.1 vs. ≤ 119.0 cm = 40.0%;P = 0.46).In contrast to hWIT, heifers with greater dRIB had greater P/AI compared with heifers with smaller dRIB (> 44.0% = 41.9 vs. ≤ 44.0% = 27.0%;P = 0.02).In addition to dRIB, another characteristic that influenced P/AI was SCFT (> 2.5mm = 44.4 vs. ≤ 2.5 mm = 23.4%;P = 0.0003), heifers with greater SCFT showed greater probability of P/AI (Fig. 7; Freitas, 2015).The zootechnics characteristics related to body development have different influence on P/AI.In the aforementioned study, hWIT and RTS isolated have no effect on pregnancy to TAI.However, age, weight, BCS, dRIB and SCFT are associated with an increase in the success of gestational establishment at TAI.When associated, the variables dRIB, age and SCFT are the characteristic that exert the most influence on P/AI (Freitas, 2015).Garcia et al. (2002) showed that body weight accounted for most of the variation associated with the onset of puberty followed by the contribution of circulating leptin concentrations, which increased during a period of 16 weeks before first ovulation.Others studies found that the onset of puberty is related to growth rate and the amount of body fat (Nogueira, 2004).In a recent study, heifers were weaned at approximately 4 month of age and fed diets to promote relatively low (0.5 kg/day) or high (1.0 kg/day) rates of body weight gain until 8.5 month of age (Alves et al., 2015).Heifers that gained body weight at a greater rate exhibited greater circulating concentrations of leptin and reduced overall NPY expression in the arcuate nucleus.The authors suggest that such changes may mediate the nutritional programming of the reproductive neuroendocrine axis and facilitate an early onset of puberty in heifers (Alves et al., 2015).
More recently, two experiments were conducted with the objective of studying the factors that affect the pregnancy rate of 14 months old Nelore heifers submitted to TAI (n = 404) and natural breeding (n = 893; bull to heifer ratio was 1 to 30; Martins et al., 2017).On both studies, presence of CL, uterine score (USC; A = uterine horns diameter > 2 cm; B = uterine horns with diameter between 1.5 and 2 cm; and C = uterine horns with diameter < 1.5 cm), diameter of the largest follicle, daily average weight gain (DAWG), loin eye area (LEA), SCFT and hWIT were analyzed at the beginning of the breeding season.On TAI experiment, none of the heifers was cycling or had an A USC on the beginning of the breeding season.The P/AI was greater on heifers with greater USC [B = 41.1% (122/297) vs. C = 17.8% (19/107); P = 0.0005].Pregnancy probability was greater for animals with greater SCFT (r 2 = 0.208; P = 0.005) and DAWG (r 2 = 0.168; P = 0.0007).The LEA (r 2 = 0.115; P = 0.13) and hWIT (r 2 = 0.309; P = 0.28) characteristics did not affect P/AI.This findings are in agreement with previous (Freitas, 2015).On natural breeding experiment, the cyclicity rate was 5.3% (47/893) at the beginning of the breeding season.Pregnancy diagnosed on day 50 of the breeding season was greater (P > 0.0001) for cycling (53.2%; 25/47) than anestrous (13.4%; 113/846) heifers and for heifers with greater uterine development [USC: A = 64.0%(16/25); B = 17.9% (116/647) and C = 3.2% (7/221); P < 0.0001].The pregnancy probability was not influenced by SCFT (r 2 = 0.096; P = 0.42) and LEA (r 2 = 0.061; P = 0.61).However, DAWG positively influenced the probability of cyclicity (r 2 = 0.263; P < 0.0001) and pregnancy (r 2 = 0.093; P = 0.005); and hWIT negatively influenced pregnancy probability (r 2 = -0.082;P = 0.03).Pregnancy probability was also increased according to diameter of the largest follicle (r 2 = 0.117; P = 0.0004).Thus, it was possible to verify that heifers with greater SCFT and DAWG had greater pregnancy probability to TAI.Heifers submitted to natural breeding with greater DAWG and diameter of the largest follicle, and smaller hWIT had greater probability to become pregnant.Further, pregnancy rates to TAI and natural breeding were greater on heifers with greater USC (Martins et al., 2017).
Thus, it is possible to obtain pregnancy rates > 70% in Nelore heifers with 14 months old after three TAI (Colli et al., 2017;Fig. 5) and that there is a positive correlation between weight, age, BCS, SCFT, and diameter of the largest follicle at the end of the synchronization protocol and P/AI 30 days after TAI.

Conclusions and future directions
The reduced reproductive efficiency of Brazilian bovine herd is still a limiting factor for the sustained growth of beef and dairy chains.We are well below the capacity of production of calves per cow per year (~68.5% weaning rate) and the quality of produced calves is far short of the ideal (~12% of females are inseminated).Besides, our heifers start reproductive life still very belatedly (around three to four years old), and reproductive efficiency is still impaired.In dairy, Brazil is not self-sufficient to supply the national market and the low productivity per cow (less than 2,000 L of milk per lactation) evidences the low genetic selection.Thus, our production system requires the development of complementary strategies to hasten and maximize the use of AI in dairy and beef herds, with easy and direct application and high reproductive efficiency to improve productivity.
In this context, several synchronization protocols (i.e. with different duration of P4 source treatment and acceleration of luteolysis) for dairy and beef cows and heifers were well studied and are shown herein.Also, the use of Resynch programs can reduce the interval between services and allow the massive use of AI in farms, resulting in the production of greater quantity and quality of calves.Finally, heifers' reproductive age can be hasten by correctly managing their mothers so they give birth in the most adequate season of the year (greater weaning weight), associated with calves' and heifers' correct nutrition and the use of adequate characteristics of evaluation as age, weight, BCS, USC, DAWG, dRIB and SCFT to select the heifers that are more suitable for early reproduction.
The dissemination of the use of these strategies isolated or in combination enables the production of greater quantity (reduction of the calving interval and hastening of reproductive age of heifers) and quality (maximization of the use of AI) of beef calves, and the production of dairy females with greater milk production per lactation (reduction of the calving interval associated to genetic gain).These technologies can contribute to improve the national production of kilograms of meat and liters of milk per hectare (what is a reflection of production efficiency), and consequently improve livestock profitability.

Figure 2 .
Figure 2. Scheme of three Resynch programs for timed artificial insemination (TAI) of beef females (based in a 90 day breeding season): 1) Traditional Resynch = treatment starts at pregnancy diagnosis (28-32 days after previous AI) in nonpregnant cows; 2) Resynch 22 = treatment starts in all cows (unknown pregnancy status) 22 days after the previous TAI and continue only in nonpregnant cows diagnosed eight days later by conventional ultrasonography (embryonic vesicle); 3) Resynch 14 = treatment stars in all cows (unknown pregnancy status) 14 days after the previous TAI and continue only in nonpregnant cows diagnosed eight days later by color Doppler ultrasonography (CL vascularization).TAI = timed artificial insemination; US = pregnancy diagnosis by conventional ultrasonography; P4 = progesterone; EB = estradiol benzoate.

Figure 3 .
Figure 3. Service rate (SR) for different Resynch programs: Resynch 14 (starts 14 days after previous timed artificial insemination (TAI), with 24 days interval between AI and SR = 87.5%),Resynch 22 (starts 22 days after previous TAI, with 32 days interval between AI and SR = 66%), and traditional Resynch (starts after pregnancy diagnosis 30 days after previous TAI, with 40 days interval between AI and SR = 52.5%).Ideal model refers to a 21 day-interval between AI and SR = 100%.

Figure 4 .
Figure 4. Pregnancy rate in Bos indicus beef cows after timed artificial insemination (TAI) with subsequent resynchronization for sequential TAI or bull exposure.Resynchronization was done using Resynch 22 program starting 22 days after the previous TAI.Groups were: 3 TAI (TAI + Resynch 22 and TAI + Resynch 22 and TAI; n = 450); 2 TAI (TAI + Resynch 22 and TAI + clean-up bulls; n = 300); 1 TAI (TAI+ clean-up bulls; n= 755).Pregnancy diagnosis of the 3 rd TAI and bull mating were done at the end of the breeding season.Adapted from Crepaldi et al., 2017.

Figure 5 .
Figure5.Pregnancy rates in young Bos indicus beef heifers (12-15 months old) after timed artificial insemination (TAI) with subsequent resynchronization using Resynch 14 (program starting 14 days after the previous TAI in all heifers, with unknown status of pregnancy).*Refers to heifers diagnosed as pregnant at first evaluation and them diagnosed as nonpregnant and reinsemination for the second time with the 3rd service heifers.Adapted fromColli et  al., 2017.

Figure 6 .
Figure 6.Scheme of two Resynch programs for timed artificial insemination (TAI) of dairy females: 1) Traditional Resynch = treatment starts at pregnancy diagnosis (32 days after previous AI) in nonpregnant cows, with 42 days interval between AI; 2) Resynch 25 = treatment starts in all cows (unknown pregnancy status) 25 days after the previous TAI and continue only in nonpregnant cows diagnosed eight days later by conventional ultrasonography (embryonic vesicle), with 35 days interval between AI.US = pregnancy diagnosis by conventional ultrasonography.

Table 1 .
Efficiency of TAI protocols with different periods of progesterone (P4) or progestin source in different categories of cattle.

Table 2 .
Efficiency of TAI protocols with three (administration of PGF on day 0 and on the days of device removal) or four animal handlings (administration of PGF two days prior to device removal) in different cattle categories.handlings with PGF treatment only on the day of P4 device removal.†New, 1x used and 2 used P4 device.

Table 3 .
Cut-off points of the evaluated parameters and calculated by the receiver operation characteristics (ROC) curve, based on the pregnancy rate.