Antral follicle count in cattle : advantages , challenges , and controversy

The antral follicle count (AFC) represents the number of follicles visualized by ultrasonography in the ovaries. Antral follicle count tends to be variable among cows but with high repeatability within the same individual. In the last decade, AFC has been considered a biological marker of fertility with many positive aspects of reproductive efficiency for those animals with high AFC. For instance, a larger number and better-quality embryos, better pregnancy rates, increased concentrations of circulating progesterone, in addition to other characteristics linked to fertility, are all responses observed in individuals with high compared with those with low AFC in Bos taurus cattle. However, the positive association between AFC and fertility did not follow the same pattern for Bos indicus. Recent articles showed no connection between fertility and number of antral follicles or better reproductive efficiency for high AFC group in Zebu cows. Thus, the aim of this review is to discuss the various data concerning AFC between indicus and taurus cattle. Additionally, we consider AFC to be a possible tool to improve cattle performance in reproductive biotechnology.


Introduction
Assisted reproductive technologies, such as artificial insemination and embryo production, represent crucial tools to improve genetic merit in cattle production (Mapletoft and Hasler, 2005;Hansen, 2014;Bó et al., 2016).Recently, reports in the literature have documented improved reproductive parameters and response to reproductive biotechnologies in cattle with increased antral follicle count (Ireland et al., 2011;Rico et al., 2012;Silva-Santos et al., 2014a).These first reports described benefits of high AFC in Bos taurus, and the evaluation of antral follicles by ultrasound was considered the most practical strategy to classify a cow for reproductive purposes.Despite the considerable variability in AFC among cows, the number of antral follicles observed in the same animal is highly repeatable over several evaluations (Burns et al., 2005;Morotti et al., 2017).This reproducibility of AFC in the same individual becomes a strategic resource for classifying an animal by the AFC with a single ultrasound examination.For taurus animals, AFC is directly correlated with the size of the ovarian follicular reserve (Ireland et al., 2011), which was not proven in indicus females when examining fetuses, heifers, and cows (Silva- Santos et al., 2011).However, other factors, such as genetics (Walsh et al., 2014), maternal environment, nutritional status, and healthiness (Ireland et al., 2011;Evans et al., 2012) also appear to influence the AFC.For example, nutritional status and general metabolism were mentioned as factors that affect follicular growth, oocyte quality, and secretion of reproductive hormones in cattle (Jimenez-Krassel et al., 2009;Mossa et al., 2010;Evans et al., 2012).
Considering Bos taurus cattle, the AFC has been directly linked to the female reproductive performance.Several reports suggest that high AFC is related to the total number of follicles that are morphologically healthy (Ireland et al., 2008(Ireland et al., , 2011)).Additionally, the number of oocytes and blastocysts (Guerreiro et al., 2014), as well as the concentration of progesterone, were greater for cows with more antral follicles (Jimenez-Krassel et al., 2009).Moreover, females with high AFC showed a greater number of embryos produced by the donors in Bos taurus (Ireland et al., 2008), crossbred indicus-taurus (Silva- Santos et al., 2014a) and Bos indicus (Santos et al., 2016).Additionally, cows with high AFC presented greater pregnancy rates compared with those with low AFC (Evans et al., 2012;Mossa et al., 2012).
In contrast, other researchers reported controversial data regarding AFC and parameters of fertility (Santos et al., 2013).Recent studies with timed artificial insemination (TAI) in indicus and crosses of indicus-taurus cattle described no positive correlation between AFC and pregnancy rates (Mendonça et al., 2013;Santos et al., 2014).Interestingly, certain results suggest better pregnancy rates for low AFC cows (Santos et al., 2013).
Considering the application of AFC as a valuable tool to assist cattle performance and reproductive biotechnology, this review aims to discuss the following: i) the relationship between the number of antral follicles and physiological parameters; ii) the challenges to applying AFC in the field, and iii) the different data for AFC between several research teams.

Repeatability of AFC and anti-müllerian hormone
Several studies performed in taurus (Burns et al., 2005;Ireland et al., 2008), indicus (Santos et al., 2012;Silva-Santos et al., 2014b) and indicus-taurus crosses (Silva- Santos et al., 2014a) reported AFC to be a parameter that was highly variable among cows but with a very consistent repeatability within the same animal.Based on this characteristic, the females can be classified as low, intermediate, or high AFC according to the number of antral follicles visualized by ultrasonography (Ireland et al., 2008;Guerreiro et al., 2014;Silva-Santos et al., 2014a).An important aspect related to AFC is its high correlation with concentrations of the anti-müllerian hormone (AMH).
The AMH belongs to the β-growth factor superfamily and is synthesized from granulosa cells of antral and preantral follicles (Cate et al., 1986).The concentration of this hormone in blood is highly correlated with the AFC.Thus, AMH can be considered an endocrine marker of the AFC in Bos indicus and Bos taurus females (Batista et al., 2014;Morotti et al., 2015).The significant advantage of this property is the possibility of using AFC instead of AMH dosage, as a single ultrasound evaluation is less expensive and easier than determining the AMH concentration.In taurus animals, a high correlation (r = 0.88, P < 0.001) was observed between the AMH concentration and the ovarian AFC.The females classified as high AFC showed greater concentrations of AMH when compared to cows with low AFC (Ireland et al., 2008).The same pattern was observed in Bos indicus, with high AFC cows presenting greater AMH concentration than cows with low AFC (Batista et al., 2014).In this context, the AMH is recognized as a reliable indicator of ovarian activity and ability to respond to a super-stimulation protocol and ovum pick-up/in vitro production (IVP; Rico et al., 2009;Baruselli et al., 2016).

Concentrations of progesterone and AFC
The production of progesterone by the corpus luteum (CL) is necessary for maintaining the appropriate uterine environment, enabling embryonic development and maintaining pregnancy in domestic animals (Bazer et al., 2010;Pohler et al., 2012).Consequently, low progesterone concentration is associated with reproductive problems, such as embryonic mortality and slower development of the endometrium in cattle (Inskeep, 2004;Diskin and Morris, 2008).
Interestingly, the number of follicles in the ovary was associated with concentrations of progesterone, as described by Martinez et al. (2016), corroborating the data of previous studies (Ireland et al., 2011;Evans et al., 2012).All these authors reported greater concentrations of plasma progesterone in taurus cows with high AFC during diestrus and pregnancy compared with low-AFC females.The reduced concentration of progesterone in plasma of low-AFC females was associated with decreased capacity of luteal and granulosal cells to produce progesterone, and reduced abundance of STAR and mRNA for STAR and LH receptor in CL (Jimenez-Krassel et al., 2009).

Antral follicle count and efficiency in embryo production
Increased AFC has been related to a larger number of IVP embryos for both Bos taurus and Bos indicus cows (Taneja et al., 2000;Singh et al., 2004;Silva-Santos et al., 2014a).Considering Bos taurus cattle from Europe, the group classified with low AFC presented smaller number of embryo compared with cows classified in the high AFC group (Ireland et al., 2007).In the same way, indicus cows with high AFC also presented a greater number of embryos than those considered to have low AFC as shown in Table 1.Considering the efficiency of embryonic production, and not only the total number of ova/embryos, the relationship between IVP and AFC in indicus donors is controversial.Several studies with indicus showed the same pattern of that obtained from taurus donors.For example, Nelore cows (n = 66) classified into high (>40 follicles), intermediate (18 to 25 follicles) or low AFC (<7 follicles) groups produced 42, 32 and 13% (P < 0.05) of total blastocyst rates, respectively (Santos et al., 2016).Considering indicustaurus heifers classified in high (≥40 follicles) or low (≤10 follicles) AFC groups, the results showed a greater number of embryos produced in females with high AFC compared with those with low AFC (6.9 ± 5.3 vs. 1.9 ± 2.1; Silva- Santos et al., 2014a).However, Monteiro et al. (2017) did not find any advantage of a high AFC when considering IVP of embryos.Those authors classified Bos indicus females into high (>15) or low (<15) cumulus-oophorous complex (COC) counts, and data were collected through 12 consecutive OPU/IVP sessions.Differences in COC and number of blastocysts were observed, but production of blastocyst per COC did not differ according to category of COC (Tab.1).
Similar results were obtained by another team.The authors studied a large number of Nelore donors (n = 356), which were classified into high (>92 follicles), intermediate (46 to 76 follicles), or low AFC groups (<31 Anim.Reprod., v.14, n.3, p.514-520, Jul./Sept.2017 follicles).In vitro embryo production did not differ among the three groups: 40, 36 and 38%, respectively (Rosa et al., 2015).Thus, although several studies involving Zebu animals demonstrate a positive correlation between embryo production and high AFC, this aspect is not entirely clear in Bos indicus cattle (Tab.2).To date, only a quantitative advantage for high AFC is evident, but proportion of blastocyst obtained per COC is not supported by indicus donors with more antral follicles.
To date, it is not possible to clarify the controversial data regarding AFC and IVP.We have several considerations to comment regarding the contradicting results between Bos taurus and Bos indicus.First, we should consider the physiological differences involving reproductive patterns between taurus and indicus cattle.Several aspects have been reported on that (e.g., number of follicular waves, metabolism of hormones, and diameter of ovulatory follicle).The influence of AFC on IVP may be another physiological difference related to breeds of European origin compared with Zebu cattle.Another question involved in the different data of AFC and IVP is the classification of animals into high, intermediate, or low AFC groups.The literature in this area is heterogenous and there is no standard to establish the follicular groups.In summary, the full understanding of how AFC could influence IVP is not understood to date.We believe that further studies will certainly help to improve our knowledge on the subject.

Fertility parameters and AFC in cows
The hypothesis that fertility is influenced by the number of follicles and oocytes in ovaries is an old idea (Hunter, 1787;Erickson, 1966).Over the years, several studies have described a high AFC as being positively correlated with female fertility.Many advantageous features that affect fertility have been related to the number of follicles in the ovary (Ireland et al., 2008), such as plasma progesterone concentration, (Jimenez-Krassel et al., 2009;Martinez et al., 2016), shorter interval between calvings (Mossa et al., 2012), and CL function and endometrial thickness (Jimenez-Krassel et al., 2009).However, it is important to emphasize that the positive correlation between AFC and cow fertility was mainly described in Bos taurus cattle (Mossa et al., 2012(Mossa et al., , 2013;;Walsh et al., 2014;Jimenez-Krassel et al., 2015;McNeel and Cushman, 2015;Santos et al., 2016).
However, even for taurus cows, the association between AFC and fertility parameters may be controversial.When considering New Zealand taurus lactating dairy cows, it was not possible to verify any association between AFC and proportion of cows pregnant after artificial insemination, as shown in Table 3 (Martinez et al., 2016), but future studies may contribute to improving the accuracy of this information.When considering studies with indicus (Nelore) and indicus-taurus (Braford), the connection between of AFC and fertility seems to be even more complex.The evaluation of follicular dynamics of Nelore cows with low AFC (<15 follicles) demonstrated greater follicular diameter at TAI (13.4 vs. 12.2 mm, P ≤ 0.05) and increased pregnancy to insemination (62 vs. 50%) than cows with a high AFC (>45 follicles; Morotti et al., 2017;unpublished data).An increased rate of follicular growth and larger follicular diameters were previously described in indicus-taurus (Santos et al., 2012) and indicus cows with low AFC (Morotti et al., 2014).A larger ovulatory follicle diameter has been clearly associated with better pregnancy per insemination (Sá Filho et al., 2010;Pfeifer et al., 2012), and this aspect reinforces the hypothesis that cows with low AFC may present increased pregnancy rates.

Relationship between AFC and phenotypic and genotypic production characteristics
A recent study was performed to verify the influence of the phenotypic and genotypic characteristics of the genetic improvement program on AFC indicus-taurus heifers.This study revealed only one parameter, the visual score for finishing precocity at weaning, that had a very small negative correlation with AFC, and all other parameters evaluated for genetic merit for beef cattle showed no correlation with AFC (Morotti et al., 2017).
Another recent study conducted in crossbred beef heifers (Bos taurus, n = 95) confirmed that AFC is associated with calving day.Heifers giving birth early in the calving season presented more antral follicles than heifers giving birth later in the calving season (McNeel and Cushman, 2015).Therefore, the AFC can be used as an ovarian phenotype for the reproductive tract in commercial production.
For dairy cattle, it was demonstrated that AFC is a reproductive characteristic with moderate heritability that is affected by age and lactation status but not correlated with milk production or other characteristics associated with the genetic merit (Walsh et al., 2014).
When considering beef and dairy cattle, the AFC seems to not be related to parameters of production, either for meat or milk.In this way, the number of antral follicles could be used as secondary criteria for considering the use of reproductive biotechnologies.The first aspect always must be the genetic merit of the dam.Next, an analysis of AFC may be considered depending on the technique to be used.

Possible mechanisms involving AFC and fertility
The physiological mechanisms regarding number of follicles and fertility parameters are not currently understood.To date, most of the published literature has described primarily applied results.In other words, most published articles focused on rates of pregnancy, embryo production, follicular growth, and similar responses; however, a thorough description of the molecular mechanisms that underlie the differences among individuals with distinct AFC remains to be elucidated.
The original articles regarding AFC pointed to several advantages for females with a larger number of antral follicles.However, a relationship with low AFC and better fertility, such as observed in recent studies with TAI in Bos indicus seems to be more easily explained.The first results about follicular dynamics and AFC have shown larger ovulatory follicles in low-AFC cows compared with those with a high AFC (Morotti el al., 2017;unpublished data).When considering fewer number of follicles to receive gonadotropin stimulation, it is possible to expect that each follicle would obtain a larger amount of FSH at the emergence of the follicular wave.Conversely, those females with a high AFC would have more follicles to share the same amount of gonadotropins.After comparing the diameter of the ovulatory follicle, many articles have described better pregnancy rates for cows with moderate to large pre-ovulatory follicle diameter compared with females with small follicle diameter at ovulation (Sá Filho et al., 2010;Pfeifer et al., 2012).In this way, the better pregnancy rates in cows with a low AFC compared with cows with a high AFC might be explained by those with low AFC having larger diameter of the ovulatory follicle.
Regarding the IVP, the majority of the studies in the literature did not present a clear advantage for any AFC category, or the studies' results are controversial.This might be explained by the artificial steps the oocytes undergo with in vitro embryo production.The nuclear and cytoplasmic maturation of oocytes are finished in vitro aside from the follicular environment.In this way, it is possible to believe that the influence of the follicle on the oocyte will be lower than in in vivo conditions, such as artificial insemination.We believe this possibility could at least partly explain the differences of AFC between artificial insemination and IVP programs.

Challenges related to AFC
The largest problem in AFC studies has been the significant variation in the evaluation criteria used to establish the categories of high, intermediate, or low counts, making it difficult to compare data (Morotti et al., 2015).For example, an intermediate category in one published paper may be considered a low or even a high category for another paper.This question becomes more critical when comparing AFC between taurus and indicus cattle.Zebu cattle usually show two, three, or even four times more follicles than female cattle of European breeds.Therefore, it is not surprising that differences in follicullogenesis between species or subspecies of domestic cattle might also affect how AFC is associated with reproduction, and extrapolation of data from Bos taurus taurus to Bos taurus indicus breeds might be incorrect and further work is warranted to identify the underlying mechanisms that explain such differences in responses.
Another critical question is the interval among AFC categories.Several articles have considered specific cut-points with only one follicle to distinguish one AFC category from another.For example, a low AFC as being fewer than 15 follicles and an intermediate AFC as being equal to or greater than 15 follicles.We believe that it is highly critical to keep the interval close, but at the same time it is easy to imagine that one follicle could be missed during an ultrasound evaluation.Therefore, we suggest a more conservative Anim.Reprod., v.14, n.3, p.514-520, Jul./Sept.2017 approach with a safety interval of AFC categories, eliminating a portion of the animals with AFC in between categories to maintain a more accurate classification of individuals.In other words, low AFC as fewer than 15 follicles and the intermediate category of AFC could start at greater than 20 or even greater than 25 follicles.The total number of animals in the experiment would be less because many cows would be discarded.Conversely, it would be possible to introduce more consistency in the AFC comparisons, because individuals with borderline values for AFC would be excluded.
Another critical challenge regarding AFC is related to the technique used.For example, females with high AFC are advantageous when submitted to OPU and IVP.For taurus donors, there are benefits regarding total numbers of embryos, as well as better rates of embryonic development.For indicus cows, it is wellaccepted that there is a better performance only on the number of embryos.However, for pregnancy rates after AI, the results between indicus and taurus cattle are notably conflicting.For taurus herds, it is wellestablished that high AFC will be associated with best pregnancy rates in AI programs.On the other hand, for Zebu females, the relationship between AFC and pregnancy per AI remains equivocal.In fact, several studies have shown improved proportion of pregnancy in cows with low or intermediate AFC (Jimenez-Krassel et al., 2017;Santos et al., 2013Santos et al., , 2016)).
Conversely, there are many factors that warrant further study, such as the relation of AFC with follicular growth, quality of oocytes, nutritional and health status of the female.Additionally, the maternal environment during gestation seems to determine the size of the ovarian reserve in their offspring (Ireland et al., 2011;Evans et al., 2012), and this aspect of maternal imprinting merits better understanding.

Final remarks
The relationship between AFC and reproductive performance represents a great challenge to the current reproductive scenario in cattle.Studies with Bos taurus herds have pointed to better reproductive performance in females with high AFC, regardless of biotechnology.However, studies conducted with Bos indicus animals have shown that AFC may be associated with performance differently according to the technique used (i.e., AI vs. IVP).Additionally, a weak correlation between genetic merit and number of antral follicle has been demonstrated.This finding may be used when considering AFC and the biotechnology to be used, as no interferences on genetic merit would be expected.We predict that AFC may be another tool for improving reproductive efficiency in cattle, perhaps something less determinant, as initially proposed years ago.However, AFC clearly remains a useful strategy for improving programs of reproductive performance in cattle.