Antral follicle count, productive and reproductive parameters in Bos indicus and Bos indicus-taurus prepubertal heifers with early puberty induction

Prepubertal Nelore (G-N = 15) and crossbred Nelore x Aberdeen Angus heifers (G-NA = 15) were used for this study. AFC, live weight, body condition score (BCS), ovary and dominant follicle (DF) diameters were determined in each animal. Puberty induction was performed by insertion of a 4 th use progesterone device (D0) which was removed on D12. Also, 1 mg estradiol benzoate was administered, and estrus intensity was classified (D12). At D21, the presence and diameter of the corpus luteum (CL) were registered. AFC was highly repeatable, regardless of hormone induction in both G-N (r=0.79) and G-NA (r=0.90). The mean AFC was greater in G-N compared to G-NA (24.2±8.5 vs. 17.7±9.0 follicles). A variation in BCS throughout the study occurred in G-NA, but not in G-N. The average weight gain (AWG) was greater in G-NA compared to G-N (0.69±0.33 vs. 0.40±0.29kg/day). The G-NA resulted in a larger diameter of DF at D12 than G-N (11.6±2.7 vs. 9.3±1.5mm). In conclusion, AFC was greater in Nelore heifers, although in both breeds this count was highly repeatable during puberty induction. Crossbred heifers had greater BCS and AWG with greater diameter of DF, indicating higher precocity when compared to Nelore heifers.


INTRODUCTION
The ability of heifers to conceive early at the beginning of the breeding season is traditionally dependent on pubertal entry and onset of the estrous cycle (Diskin and Kenny, 2014).Studies look for ways to induce early puberty in heifers aiming a reduction of age at first calving, an increase of reproductive efficiency and genetic gain, which is an important goal to increase production per area and animal production capacity (Sá Filho et al., 2013).In this context, studies have investigated the relationship between antral follicle count (AFC) and reproductive performance in heifers and cows since AFC is related to female fertility (Ireland et al., 2007;Mossa et al., 2012;Morotti et al., 2017aMorotti et al., , 2018;;Zangirolamo et al., 2018;Seneda et al. 2019).
The AFC is a remarkable ovarian characteristic in cattle, being highly variable among females, but highly repeatable in the same individual (Burns et al., 2005;Ireland et al., 2007).AFC can be defined as the estimated number of antral follicles with diameter ≥3mm during a follicular wave of estrous cycle.Therefore, through an ultrasound examination of the ovaries, it is possible to count all antral follicles and classify females in low, intermediate, or high AFC (Burns et al., 2005;Ireland et al., 2007;Morotti et al., 2018;Moraes et al., 2019).North American and European studies evaluated the correlation of AFC with the reproductive performance of Bos taurus taurus cows and concluded that high AFC show better reproductive and productive performance (Ireland et al., 2011;Evans et al., 2012) as well as studies have stated that low AFC demonstrated lower fertility (Mossa et al., 2012).
In contrast, studies performed with Bos indicus cattle in Brazil has revealed controversial data to those found in Bos taurus (Santos et al., 2016;Morotti et al., 2018;Moraes et al., 2019;Lima et al., 2020).For example, females with high AFC have better performance for in vitro embryo production; but had no better pregnancy rate to timed artificial insemination TAI (Santos et al., 2016).In addition, recent studies (Morotti et al., 2017a;2018;Moraes et al., 2019;Seneda et al., 2019;Lima et al., 2020) have shown a positive influence of AFC on ovarian follicular dynamics and reproductive performance of Nelore females, contrary to the results found in taurine herds outside Brazil.
Crossbreeding heifers (F1; Bos indicus-taurus) have often been kept on the farm as a future breeder (Cardoso et al., 2018).In this context it is important to highlight that there are important reproductive differences between the subspecies (Sartori and Barros, 2011).In addition, there is evidence that suggests that the total number of ovarian follicles may influence response to hormone synchronization protocols (McNeel and Cushman, 2015) and in beef heifers, AFC can be positively associated with entry into puberty.Nevertheless, the relationship between AFC and reproductive performance in heifers is limited, especially to association with puberty induction.
Therefore, we hypothesized that even in animals as young as in this study, AFC is highly repeatable in both Bos indicus and Bos indicustaurus heifers throughout the puberty induction protocol.In addition, we tested the hypothesis that animals with taurine blood show favorable results for productive aspects and sexual precocity.Therefore, this study aimed to evaluate the repeatability of AFC and to compare the productive and reproductive parameters in Bos indicus and Bos indicus-taurus heifers submitted to early puberty induction

MATERIAL AND METHODS
The present study was conducted according to the standards of the Ethics Committee for Animal Experimentation of the University of Londrina, based on Federal Law 11.794 of October 8, 2008, andwas approved under number CEUA 19514.2018.86.For this study a group (G) of pre-pubertal heifers (n=30), Nelore (G-N; n=15; Bos indicus) and Crossbred (Nelore x Angus; G-NA; n=15; Bos indicus-taurus) were evaluated to determine the number of antral follicle/AFC, body weight and body condition score (BCS) 15 days before the initiation of the puberty induction protocol (Day -15).The pre-pubertal condition was confirmed in two ultrasound exams (Day-15 and Day 0) and only heifers without CL and with follicles smaller than 8 mm in diameter were kept in the study.The animals from both groups were kept together in an area composed of Brachiaria brizantha cv marandu (15% of the area),
The live weight (kg) was measured by a scale without prior fasting.BCS was assessed using a scale from 1 to 5 as described by Machado et al. (2008).Each heifer was evaluated by transrectal palpation, followed by ultrasonography for uterine and ovarian examination.All evaluations were performed by a single veterinarian on Days -15, 0, 12 and 21, being that all variables were monitored on each assessment day.
Fifteen days before (Day -15) the initiation of the puberty induction protocol, each animal was evaluated only once for AFC estimation.During this examination, each ovary (right and left) was observed in detail with an ultrasound (SonoScape® S2 equipped with a 5Mhz rectal linear frequency transducer) and all visible follicles (follicles≥3mm) were identified and counted as described by Morotti et al. (2018).Briefly, the entire ovarian surface was scanned starting from one face close to the pedicle to the other face.AFC was defined by the sum of follicles from both ovaries.Diameter of dominant follicle was calculated from two crosssectional linear measurements of the follicular antrum captured on the ultrasound monitor.Ovary diameter was obtained from largest image of an ovary captured on the monitor, and the measurements of each ovary image were determined.The animal identification, follicle numbers, follicle diameters, ovulation status, BCS, and weight were recorded on individual data sheets.
Weight, BCS and ovarian assessments were performed on Days -15, 0, 12 and 21.The heifers received a puberty induction protocol on Day 0, as described in Fig. 1.Hormone treatment was performed as suggested by González et al. (2020) that consisted of the insertion of a 4 th use intravaginal progesterone (P4) device (CIDR®, Zoetis, Sao Paulo, Brazil) that remained until day 12, when the P4 source was removed.On the same day of the device withdrawal, 1mg estradiol benzoate (EB; Gonadiol®, Zoetis, Sao Paulo, Brazil) was applied in addition to the fixation of an adhesive (Estrotect ™, Spring Valley, United States) in the sacrococcygeal region to evaluate the estrus expression rate and perform the heat intensity classification (0 = absent, 1 = low and 2 = high intensity).The heat intensity was defined by the amount of the grey coating that was removed from the fixing adhesive similar to what was described by Nogueira et al. (2019).
To confirm ovulation, the heifers were evaluated between 48 and 60 hours after ovulation induction and the absence of the dominant follicle used to indicate that ovulation had occurred and reconfirmed by the presence of the corpus luteum (CL) in the same ovary on the 21st day after the onset of the puberty induction protocol.
Quantitative variables were analyzed by the generalized linear model (GLM), including the main effect of the genetic grouping as a fixed effect and all other sources of variation were included as covariates of the model.In the case of repeated variables, data were analyzed by repeated measures ANOVA using GLM, including the time effect as the main random effect and the other sources of variation as covariates.
In the presence of a significant effect, the averages were compared by the Tukey test.Differences in ovulation rate between genetic groups were analyzed by Fisher's exact test.Repeatability (range 0 -1, where 1 = perfect) was defined as the proportion of the total variance that could be attributed to the animal variation and was calculated as follows: ẟ2 animal/(ẟ2 animal + ẟ2 error).
For descriptive analysis, quantitative data are presented as mean (M) and standard deviation (SD) and qualitative data as percentage (%).All statistical analyzes were performed using the Minitab® statistical software version 18.1.To indicate an effect of categorical variables and their interactions, a significance level ≤ 0.05 was adopted.

RESULTS AND DISCUSSION
Comparing Nelore (Bos taurus indicus) and Nelore x Angus (Bos indicus-taurus) heifers, AFC was highly repeatable during the study days and regardless of hormonal induction, both in G-N (r=0.79) and G-NA (r=0.90).The AFC proved to be a reproductive trait highly consistent in both genetic groups, being more constant in crossbred heifers than in Nelore.However, AFC was greater (P < 0.05) in G-N compared to G-NA at Day 0, 12 and 21, except for Day -15, as shown in Table 1.Furthermore, the BCS varied (P<0.001) in G-NA and was greater (P<0.05) in G-NA than G-N.In this study the AFC was highly repeatable between genetic groups and highly constant among the evaluated heifers, proving to be a reliable evaluation parameter.The number of antral follicles was greater in Nelore than crossbred heifers.However, the dominant follicle from crossbred animals showed a larger diameter on the day that the P4 device was removed, indicating a better response to the puberty induction protocol of this genetic group.In addition, Nelore x Angus heifers also showed a better productivity parameter, resulting in superior weight gain and BCS during all days of the experiment.The innovative and determining factor of this study was the precocity of the studied animals, since there are few studies comparing the reproductive and productive parameters in Nelore and Nelore x Angus prepubertal heifers.
Regarding physiological aspects, ovarian functions are regulated by endocrine hormones secreted by the hypothalamus (gonadotropinreleasing hormone -GnRH), anterior pituitary (follicle stimulating hormone -FSH and luteinizing hormone -LH), ovaries (progesterone, estradiol, and inhibin) and uterus (prostaglandin F2α -PGF 2α ) (Aungier et al., 2015).Elevated concentrations of estradiol secreted by the pre-ovulatory follicle in turn promote a GnRH surge and allowwhen progesterone levels are lowthe expression of behavioral estrus and the release of LH to cause ovulation.
Classically, a prepubertal heifers reach puberty when age and weight are satisfactory, being characterized by classical estrus signs, ovulation occurrence followed by luteal phase (Perry, 2016).Estrus behavior is characterized by mounting or attempting to mount other cattle, standing to be mounted by other cattle, smelling other females, trailing other females, bell owing, depressed appetite, nervous and excitable behavior, roughed up tail hair, vulva swelling and reddening and clear vaginal mucous discharge (Reith and Roy, 2017).
First ovulation is usually characterized by early regression of the CL because the heifer is not yet sexually mature (Garverick et al., 1992).In the present study, a progesterone device was used to induce puberty, since exposure to progesterone allows the secretion of LH at the end of the treatment, inducing ovulation of the dominant follicle and contributing to the formation of functional CL in the first estrous cycle (Vrisman et al., 2018;González et al., 2020).Physiologically, in prepubertal heifers, the low concentration of estradiol produced in the ovaries due to an intense negative feedback mechanism prevents the hypothalamic preovulatory center from secreting adequate GnRH pulses (Amstalden et al., 2011).Therefore, this process also inhibits the release of FSH and LH from the anterior pituitary, causing absence of follicular waves and ovulation.On the other hand, when the hypothalamus initiates GnRH neuron function (influenced primarily by nutritional control), there is decreased negative feedback from estradiol, which leads to an increase in the frequency of GnRH pulse release (Kinder et al., 1995;Amstalden et al., 2011).Thus, the treatment with exogenous progesterone reduces negative feedback caused by estradiol receptors in the hypothalamus when they are in excess, increasing the release of gonadotropins (FSH and LH) by the pituitary gland and, consequently, stimulating the growth rate and ovulation of the dominant follicle (Vrisman et al., 2018).
The response to cyclicity induction in the present study was low in both groups (Table 3).In this context, two main aspects that may justify this low response are suggested.First, it should be noted that the heifers used are very young (12 to 14 months), which may suggest the absence of minimum sexual maturity for the hypothalamuspituitary-gonad axis to be responsive to hormonal treatment (Sá Filho et al., 2015).Second, it can be hypothesized that the residual amount of P4 in the 4th use device had suboptimal concentrations, which may have compromised LH secretion at the end of cyclic induction hormone treatment (Vrisman et al., 2018), as well as Brunoro et al. (2017) presented in their study, where pregnancy rates decreased by reusing P4 devices.
In addition to the effect of P4, other substances and hormones may influence the pattern of LH secretion.The study by Macedo et al. (2019), who evaluated the effect of intramuscular administration of 25 different doses of kisspeptin (Kp) on LH release pattern in pre-pubertal Bos taurus and Bos indicus heifers, demonstrated that treatment was more efficient in Bos taurus that presented higher LH release.This shows that animals of taurine origin respond more efficiently to puberty induction protocols, as well as the crossbred heifers of the present study, which were found to be earlier than zebu heifers.
The AFC, a reproductive parameter that has been widely studied, was analyzed in this study to assess its influence on the puberty of prepubertal heifers.The mechanisms involving AFC and fertility in bovine females are not yet fully elucidated and there are challenges regarding their influence on the biotechnology used, pregnancy rate, oocyte quality, embryonic development, and in vivo and in vitro embryo production (Ireland et al., 2011;Evans et al., 2012;Morotti et al., 2017aMorotti et al., , 2017b)).However, AFC already proves to be promising as a marker of reproductive activity and productive efficiency in beef herds (McNeel and Cushman, 2015;Seneda et al., 2019).
A study performed by McNeel and Cushman (2015) confirmed that AFC is related to the time of conception of cows, and those who give birth at the beginning of the breeding season have more antral follicles.Thus, the ovarian characteristics of beef cattle females can be used as parameters for commercial meat production (Morotti et al., 2017b).For example, the high AFC seem to respond better to embryo production biotechnology (in vivo and in vitro) (Silva-Santos et al., 2014;Santos et al., 2016;Morotti et al., 2017aMorotti et al., , 2017b;;Garcia et al., 2020), although the low AFC seems to be more efficient in the TAI technique (Morotti et al., 2018;Moraes et al., 2019;Lima et al., 2020).
As is well known, AFC is greater in Nelore females (Pontes et al., 2011) and the present study confirmed this fact in prepuberal and pubertal heifers, even with a limited number of animals studied.During the hormonal induction protocol, AFC remained highly constant because of its high repeatability in both Nelore and crossbred heifer group, being a reliable marker for evaluating reproductive performance (Ireland et al., 2007;Santos et al., 2016;Morotti et al., 2017b).In this context, the present study proving that a single ultrasound evaluation would be reliable for determining the AFC of prepubertal heifers, as has been reported by Burns et al. (2005)  Throughout the puberty induction protocol of prepubertal heifers, a highest productive performance of crossbred (Nelore x Angus) heifers was remarkable, both in BCS and average weight gain evaluation.The explanation for crossbreed animals having shown a better performance is due to the relationship between puberty and body condition (Day and Nogueira, 2013) and possibly the inherent characteristics of the subspecies that tend to be more precocious in taurine herds than indicus (Sartori and Barros, 2011).However, the nutrition has been identified as one of the factors that positively influence the fully functioning of the hypothalamus-pituitarygonad axis.On the other hand, heifers with unfavorable genetic merit failed to reach puberty early, even when receiving adequate nutritional supplementation (Ferraz et al., 2018).
In addition, the diameter of dominant follicle from crossbred females was greater than the Nelore heifers, showing that in the condition of super early induction, as in the present study, crossbred animals can respond better reproductively possibly due to their sexual precocity and superior productive performance, similarly to the study shown by Paim et al. (2018).In this context, the data of the present study highlights the importance of using the crossbreed as a matrix, contributing to the beef production chain, precisely due to the advantages related to heterosis coming from the crossbreeding of the Nelore and Angus breeds.
It is important to emphasize that the relationship between AFC, reproductive characteristics and genetic merit is an essential selection factor in beef cattle herds (Morotti et al., 2017b), but little is known, and few studies have been conducted on puberty induction in prepubertal Bos taurus indicus animals relating the precocity factor, genetic merit, and AFC.Furthermore, we predict that AFC may be another tool for improving reproductive efficiency in cattle in a puberty induction protocol, especially when genetics and parameters related to puberty in heifers are also considered.

CONCLUSION
The super-early induction of puberty resulted in low cyclicity rate in Bos indicus (Nelore) and crossbred Bos indicus-taurus (Nelore x Angus) heifers.AFC was greater in Nelore heifers, although in both groups this count was highly repeatable throughout puberty induction.Crossbred heifers, however, had greater body condition score and average weight gain with greater dominant follicle diameter, indicating earlier precocity when compared to Nelore heifers.

Table 3 .
Mean and standard deviation of estrus intensity, ovulatory rate, and corpus luteum (CL) diameter in Nelore and crossbred heifers (Nelore x Angus) after hormonal protocol for puberty induction