Graduate Student Literature Review: Effects of human chorionic gonadotropin on follicular and luteal dynamics and fertility in cattle

Circulating progesterone concentrations during the growth of the ovulatory follicle and early embryo development have been positively associated with embryo quality and survival and pregnancy success. As a potent luteotropic agent with LH-like activity, human chorionic gonadotropin (hCG) has been tested in different studies to improve pregnancy outcomes by increasing circulating progesterone concentrations during the growth of the ovulatory follicle or early embryonic development. Nevertheless, hCG has produced inconsistent, contradictory, and intriguing results. Furthermore, recent research indicates that hCG, when used before artificial insemination, may affect physiological events necessary for the ovulation of a viable oocyte. In addition, the use of hCG-inducing accessory corpus luteum during the estrous cycle seems to disturb luteolysis and follicle and luteal dynamics during the estrous cycle. This literature review discusses past and current research exploring the effects of hCG on the estrous cycle characteristics and pregnancy per artificial insemination and embryo transfer.


INTRODUCTION
Reproduction performance of US lactating dairy cows suffered a period of subfertility, which was evident by a decline in daughter pregnancy rate from the 1960s and a nadir in the late 1990s (Fricke and Wiltbank, 2022).The decline in fertility has been associated with a steady increase in milk production per cow in the same period; however, daughter pregnancy rate has risen abruptly since the 2000s, even with the continuous increase in average milk production per cow in the United States.This quick and significant improvement in the fertility of lactating dairy cows has been related primarily to new technologies developed to improve fertility by controlling the hormonal milieu, follicle and corpus luteum development, and time of ovulation and insemination (Fricke and Wiltbank, 2022).Moreover, the improvement in US lactating dairy cow reproductive performance has also been associated with the achievements in dairy cattle physiology, nutrition, management, genetics, economics, herd health, and production medicine, and their integration (Norman et al., 2009).
Two specific technologies that allowed significant progress in understanding the physiology of the estrous cycle in cattle, especially ovarian function during the estrous cycle, were the use of sensitive hormonal assays and ultrasonography.The hormone-based programs that manipulate the estrous cycle and allow timed AI (TAI) were developed based on the insights related to ovarian development and the dynamics of circulating hormones during the estrous cycle.However, a limited number of commercial products are approved to manipulate reproductive function in cattle in the United States, including PGF 2α , GnRH, progesterone (P4), and human chorionic gonadotropin (hCG).Therefore, to effectively use this inventory, a thorough investigation of the effects of each pharmaceutical agent and their combination on bovine reproductive physiology is necessary to evaluate their practical use as fertility treatments.This review aims to provide current insights on the effects of hCG on the physiology of the estrous cycle and fertility parameters in cattle, combined with a historical background on the pharmacological properties of hCG.

HUMAN CHORIONIC GONADOTROPIN: THE DISCOVERY, FUNCTIONS, AND CLINICAL USE IN HUMANS AND CATTLE
The first report of hCG was by Ascheim and Zondek (1927) in Germany, demonstrating that the blood and urine of pregnant women contained a substance that, Graduate Student Literature Review: Effects of human chorionic gonadotropin on follicular and luteal dynamics and fertility in cattle* when injected into immature female mice, produced follicular maturation and luteinization.The gonad-stimulating substance named hCG is mainly synthesized by placental syncytiotrophoblast cells, but it can also be secreted by the early developing embryo and some tumors (Stenman et al., 2006;de Medeiros and Norman, 2009).In humans, hCG represents an essential embryonic signal for establishing and maintaining pregnancy by stimulating corpus luteum (CL) to secrete P4, promoting angiogenesis in the uterine endothelium (Zygmunt et al., 2002), maintaining myometrial quiescence (Ambrus and Rao, 1994), and fostering immunomodulation at the maternal-fetal interface (Schumacher et al., 2013).Clinical measurement of hCG in women is used in a variety of situations, such as diagnosis of pregnancy, pregnancy-related disorders, and gynecological cancers, and prenatal screening (Stenman et al., 2006).Administration of hCG in women is used in assisted reproductive techniques to trigger final follicular maturation and ovulation and luteal phase support.In men, hCG administration stimulates testosterone production by the Leydig cells in cases of hormone deficiency or hypogonadism (Lunenfeld et al., 2019).Most of the circulating hCG is metabolized by the liver, and approximately 20% is excreted by the kidneys (Nisula et al., 1989).In cattle, hCG has been commercialized in the United States by the name of Chorulon (Merk Animal Health) for at least 20 years.Its label use in cattle is indicated for treatment of nymphomania (frequent or constant estrus behavior) due to cystic ovaries, by an intramuscular injection of 10,000 IU of chorionic gonadotropin.However, the majority of hCG use in cattle seems to be related to inducing ovulation in other situations to stimulate P4 production by the CL (Niswender et al., 2000) or induce accessory CL formation.

HUMORAL IMMUNE RESPONSE TO hCG IN CATTLE
The high molecular weight and carbohydrate content of hCG (~38,000 Da) make it an immunogenic molecule so that repeated administration may lead to the development of antibodies against hCG in cattle ( Sundby and Torjesen, 1978;Johnson et al., 1988).Giordano et al. (2012) administered 3 doses of 2,000 IU of hCG i.m. 35 d apart in lactating Holstein cows, observing 4, 47, and 73% of antibodies against hCG, respectively.Interestingly, despite cows being exposed to hCG 3 times within 70 d in that study, some cows did not develop a humoral response, similar to other studies performed in bulls using 750 IU of hCG (Sundby and Torjesen, 1978) and mares using 2,500 IU of hCG i.v.(Siddiqui et al., 2009).In addition, the minimum level of antibodies necessary to bind all the hCG after treatment and whether the antibodies could inhibit or decrease ovulatory response after repeated exposure to hCG is still unknown.

MOLECULAR ASPECTS OF HCG AND DIFFERENCES TO LH
Human chorionic gonadotropin is part of the glycoprotein hormone family, including FSH, LH, and thyroid stimulating hormone.All glycoprotein hormones in this family are composed of 2 subunits, α and β.The α subunit is similar among all these hormones, and the β structure of hCG is 80 to 85% similar to the LH structure, providing hCG the capacity to attach to LH receptors (LHCGR, Figure 1) and exert an LH-like activity (Al-Masoody and Al-Obaidi, 2020).In addition to the similarity with LH, hCG has a longer halflife and higher binding affinity for LHCGR compared with LH (Chenault et al., 1990).Mock and Niswender (1983) observed in ovine luteal cells that the time for internalization and degradation of radiolabeled hCG bound to the LH receptor was significantly extended (22.8 ± 2.3 h) when compared with ovine LH (0.4 ± 0.2 h).The internalization of the LHCGR bound to hCG or LH is the mechanism used by the luteal cell to terminate the response to the hormone (Niswender et al., 1985).In dairy cows treated with hCG, Nascimento et al. (2013) reported increased concentrations of hCG 2 h after treatment, with maximal concentrations at 4 h and a plateau from 4 to 12 h after treatment, returning to baseline concentrations by 72 h (not different from time 0).In contrast to these results, the length of the LH surge elicited by the injection of GnRH-agonist (buserelin, 10 µg, i.m) lasts 5 h in heifers (Chenault et al., 1990), and a natural or PGF 2α -associated preovulatory surge of LH lasts approximately 10 h (Chenault et al., 1975(Chenault et al., , 1976)).Thus, hCG seems to be one of the most potent drugs to induce ovulation in cattle and as a luteotropic agonist.
Although it is traditionally believed that LH and hCG are biologically equivalent because they act via the same receptor (LHCGR), in vitro studies showed that LH and hCG are not equivalent in terms of biopotency, response kinetics, and molecular effects on human granulosa cell cultures (Casarini et al., 2012).Differences in signaling pathways between hCG and LH have been reported.For instance, hCG induced a greater activation of the cyclic AMP-protein kinase pathway, which stimulates P4 production in granulosa cells (Casarini et al., 2012).However, LH had increased potency on AKT-and ERK1/2-pathways (Ascoli et al., 2002;Casarini et al., 2012), which are involved in Cunha and Martins: LITERATURE REVIEW: EFFECTS OF hCG ON THE ESTROUS CYCLE events such as proliferation, differentiation, and survival of granulosa cells (Ben-Ami et al., 2009).These are the main pathways necessary for the activation and mediation of the LHCGR effects.Noteworthy, these results come from in vitro studies performed in human cell cultures, and the application into bovine granulosa cells requires further investigation.

FOLLICULAR AND LUTEAL RESPONSES TO HCG
Human chorionic gonadotropin induces ovulation by mimicking the LH surge necessary to ovulate a dominant follicle (DF).Although hCG does not depend on the ovarian-pituitary axis to cause ovulation, a selected DF with LH receptors in granulosa cells is necessary for ovulation to occur.The protein (Bodensteiner et al., 1996) or expression of mRNA (Xu et al., 1995;Bao et al., 1997) encoding LHCGR (follicular selection) are not expressed in the granulosa cells of growing follicles during the first 2 d of the follicular wave (d 0 = follicles ≥4 mm).Therefore, the administration of hCG is not effective in inducing ovulation during the early stages of follicle development.Price and Webb (1989) found a greater ovulatory response in heifers treated with hCG between 4 and 7 d of the estrous cycle than other stages of the cycle.
Several studies used hCG to induce ovulation of the first wave DF, aiming to form an accessory CL during the luteal phase and increase circulatory P4 concentrations (Fricke et al., 1993;Cunha et al., 2021a).This outcome was successfully achieved when hCG was administered between d 5 and 9 of the estrous cycle.Schmitt et al. (1996) induced nonlactating Holstein cows (n = 4) to ovulate on d 5 of the estrous cycle with 3,000 IU of hCG (1,000 IU i.v., and 2,000 IU i.m.), with subsequent accessory CL formation and a significant increase in P4 from d 6 to 13 of the cycle when compared with control cows.In the same study, the accessory CL from the hCG-treated group (approximately 7 d old) was removed by flank laparotomy on d 13 of the cycle, whereas the original CL was maintained.From d 14 (24 h after removal of the hCG-induced CL) to d 17 of the estrous cycle, there was no effect of treatment or interaction day by treatment on circulating P4 concentrations between groups, suggesting that the accessory CL increased circulating P4 concentrations from d 6 to 13 of the estrous cycle.
Apart from the known luteotropic effect of hCG on creating new accessory CL, hCG is also reported to potentially increase the size and volume of the original CL (Santos et al., 2001).In the study conducted by Santos et al. (2001), 406 high producing lactating Holstein cows were randomized to receive either hCG (3,300 IU) or saline treatments on d 5 after AI.Ultrasound examination of ovaries from cows on d 14 after AI showed that the surface area and volume of the largest luteal structure (assumed to be the original CL) was greater for the hCG-treated group, suggesting that the treatment with hCG may also have increased the size of the CL originated from the spontaneous ovulation.Attributed effects of hCG on original CL included an increased size of luteal cells (Schmitt et al., 1996) and a reduction in the ratio of small to large luteal cells by d 10 in ewes treated with hCG on d 5 and 7.5 of the cycle (Farin et al., 1988).It has been also speculated that the longer binding time of hCG to the LHCGR may be responsible for these effects on the original CL.However, in the study conducted by Schmitt et al. (1996), after the surgical removal of the hCG-induced CL, there were no differences in circulating P4 and luteal volume of the original CL between the control and hCG-treated cows during d 14 to 17 of the estrous cycle.These results led to speculations about the absence of an hCG effect on the original CL or a transitory effect that probably lasted for less than 7 d.Results from Cunha et al. (2021b) suggested a transitory effect of hCG on original CL volume, which increased up to 50% from d 10 to 14 of the estrous cycle when cows were treated on d 7. Still, on the day of onset of luteolysis, the original CL was about the same volume as in control cows.In the same study, another treatment group of cows received hCG on d 7 and 13 of the estrous cycle; these cows maintained the original CL larger than control through the entire cycle and even for a few days after the onset of functional luteolysis.Fricke et al. (1993) paradoxically found that a ~13-dold original CL that was exposed to 1,500 IU of hCG on d 6 of the estrous cycle (n = 5) exhibited reduced basal and LH-induced P4 secretion in vitro compared with luteal tissue from a 13 d old original CL of salinetreated control cows.However, in the same study, hCG treatment on d 6 of the estrous cycle induced the formation of an accessory CL and increased P4 circulating concentrations in vivo from d 9 to 13.In summary, the results from most studies indicated that hCG is capable of increasing P4 secretion not only due to the formation of an accessory CL but also due to a direct effect on luteal cells, which appears to be dose-dependent and temporal.

EFFECTS OF HCG ON THE ESTROUS CYCLE
Estrous cycle length has been reported to increase after treatment with hCG on d 5 to 7 of the estrous cycle (Cunha et al., 2021a,b), most likely due to a higher incidence of cows with 3 follicular wave estrous cycle (Cunha et al., 2021b).Estrous cycles with 3 waves have been determined to be longer than 2 wave cycles (Bleach et al., 2004).Interestingly, the 3-wave estrous cycle is also known for the prolonged luteal phase with later onset of luteolysis, occurring approximately 2 d later compared with the 2-wave cycle (Townson et al., 2002;Sartori et al., 2004).It seems that both circulating concentrations of estradiol (E2) produced by the DF and the endometrial E2 responsiveness are the main factors that may initiate upregulation of oxytocin receptors and timing of luteolysis (Araujo et al., 2009;Domingues et al., 2020).In cycles with 3 follicular waves, the second wave DF loses dominance and E2 secretory capacity before estradiol-α receptors could be activated, making necessary the third wave DF to grow and produce enough E2 to trigger the luteolysis onset.Most hCG treatments from d 5 to 7 of the estrous cycle cause ovulation of a DF and formation of an accessory CL, which induce an earlier second follicular wave emergence.As a result, the second wave DF becomes atretic earlier, causing a third follicular wave to emerge and a later onset of luteolysis.Recent data from our laboratory corroborates this hypothesis.Cows treated with hCG on d 7 had earlier emergence of the second follicular wave (d 8.6 vs. 12.1) and later luteolysis onset (d 19.5 vs. 18.0) in the estrous cycle compared with control cows that did not receive any treatment (Cunha et al., 2021b).When only cows with 3 waves were analyzed, the day of luteolysis onset was not different between cows treated with hCG on d 7 and control cows, suggesting that the increase in estrous cycle length by hCG was a result of a third wave induction.
In addition, prolongment of the luteolysis process increased the proportion of irregular estrous cycles (Cunha et al., 2021b), and a decrease in the proportion of multiparous cows detected in estrus by activ-ity monitor after treatment with hCG (Cunha et al., 2021a) could be related to an increase in estrous cycle length.Thus, these factors need to be evaluated in future studies investigating the use of hCG as a tool to improve fertility, especially in farms that rely on detection of estrus.

hCG VERSUS GnRH: MECHANISM OF ACTION AND THEIR FOLLICULAR AND LUTEAL EFFECTS
Both hCG and GnRH treatments are aimed to induce the same effects on the ovary by inducing ovulation of a DF (Stevenson et al., 2007).Although GnRH acts to stimulate the anterior pituitary to release LH, the mechanism of action of hCG is independent of the hypophysis by directly binding to LHCGR on the granulosa cells of the DF (Figure 2; Bodensteiner et al., 1996;Motta et al., 2020).The ovulatory response to 2,000, 2,500, and 3,300 IU of hCG has been reported to be greater compared with 100 µg of GnRH analog gonadorelin acetate.This result has been attributed to the attenuation of the GnRH-induced LH (Cabrera et al., 2021a) surge magnitude by high circulating P4 concentrations at the time of treatment (Colazo et al., 2008;Giordano et al., 2012).Therefore, the ovulatory response to hCG did not seem to be affected by systemic P4 concentrations at the time of treatment, and it was tested as an alternative strategy to increase the ovulatory response to the first treatment of the Ovsynch for lactating dairy cows compared with analogs of GnRH busereline acetate and gonadorelin acetate (Keskin et al., 2010;Cabrera et al., 2021b).
Time from treatment to ovulation may vary among GnRH (gonadorelin) and hCG, depending on the estrous cycle phase when the treatment is administered (Liu et al., 2019;Cabrera et al., 2021b).However, regardless of the estrous cycle phase, cows treated with hCG ovulate later than cows treated with GnRH (gonadorelin; Liu et al., 2019).In both studies, the difference in time for ovulation between hCG and GnRH is between 2 and 5 h.It was speculated that the ovulation might occur later after hCG treatment than GnRH because of the delayed time to hCG or LH peak concentration, which is reached 4 h after treatment with hCG (Nascimento et al., 2013) versus 1 to 2 h after treatment with 4 different GnRH analogs (Souza et al., 2009;Giordano et al., 2012).It may also be hypothesized that the differences in ovulatory responses occur because therapeutic doses of hCG and GnRH (regardless of the molecule) were not confirmed to be equipotent.
Several studies have used different doses of hCG to induce ovulation, ranging from 1,000 IU to 3,300 IU (Nascimento et al., 2013;Besbaci et al., 2020).Cabrera  2021a) found that 1,000 IU of hCG induced a similar ovulatory response (~78%) compared with 100 µg of GnRH (gonadorelin acetate) in lactating dairy cows on d 7 of the estrous cycle.In contrast, treatment with 2,500 and 3,300 IU of hCG resulted in a high ovulatory response (~94%).Hence, the authors consider 2,500 IU of hCG to be the most cost-effective dose.
It also has been reported that the CL induced by hCG produces more P4 than CL induced by GnRH.Furthermore, P4 concentrations in the culture medium of slices of CL incubated in vitro were higher in hCGinduced CL than for the GnRH-agonist-induced CL (buserelein) for all doses of LH (Schmitt et al., 1996).Hence, it appears that the increase in circulating P4 concentrations in cows treated with hCG compared with GnRH may also be due to the prolonged LH-like activity of hCG that acts on the original CL in vivo.

hCG USE BEFORE INSEMINATION
The use of TAI programs aims to control follicular and luteal development so a viable oocyte can be maturated and released in a synchronized manner, allowing programmed AI in dairy farms with no need for detection of estrus.In TAI programs, a series of hormonal treatments are administered to synchronize the time of ovulation just after the TAI.All treatments in the program are essential steps for the successful synchronization of the cows.For example, within the Ovsynch protocol (Pursley et al., 1995), cows that ovulated to the first GnRH of the program (G1) had increased response to the remaining treatments of the protocol (Vasconcelos et al., 1999;Bello et al., 2006) and greater pregnancy per AI (P/AI) when compared with cows that did not ovulate to G1 (Chebel et al., 2006;Giordano et al., 2013;Bisinotto et al., 2015).Therefore, ovulation to the G1 is a key factor that determines the success of Ovsynch.Moreover, the presence of a CL at G1 and higher P4 concentrations at PGF 2α treatment of Ovsynch were also associated with greater P/AI than cows with low P4 (Martins et al., 2011;Bisinotto et al., 2014;Wiltbank et al., 2014).
Given the importance of each treatment for the success of a TAI program, several strategies have been evaluated to increase the proportion of cows that ovulate to G1.Because of the attenuated effect of P4 on GnRH-induced LH surge mentioned before, a hypothesized alternative strategy to increase ovulatory response at the initiation of Ovsynch protocol was to replace GnRH with hCG.However, several studies rejected this hypothesis (Burns et al., 2008;Keskin et al., 2010;Cabrera et al., 2021b).These studies have reported decreased P/AI when compared with GnRH (gonadorelin or buserelin).In an attempt to understand the causes behind this unexpected decrease in fertility, Cabrera et al. (2021b) evaluated the rate of luteolysis after PGF 2α treatment in cows with accessory CL induced by hCG versus GnRH (gonadorelin) and did not find differences.Authors speculate that the long halflife of hCG in circulation and, therefore, a long LH-like activity might negatively affect oocyte quality during the growth of the preovulatory follicular wave, leading to an abnormal endocrine milieu for oocyte development.However, this hypothesis still needs to be tested.
A strategy that may improve lactating dairy cow fertility would be to increase P4 during the growth of the DF of a natural estrous cycle with insemination occurring on the following estrus event.This strategy would benefit herds that do not use any TAI protocol and solely inseminate cows after estrus detection or herds that rely on estrus detection to re-inseminate cows that failed to be pregnant after TAI.Noteworthy, activity monitors have increased in popularity in the last decade.One of the main features of these devices is to identify cows and heifers in estrus, reducing the necessity of synchronization of ovulation protocols.Despite the extensive use of TAI in US dairy farms, insemination after detection of estrus remains a common and essential breeding strategy, especially for second and subsequent AI on dairy farms (Caraviello et al., 2006;Van Schyndel et al., 2019).However, very few studies aimed to investigate strategies to improve the fertility of cows inseminated following the detection of estrus, which seems to remain the same over the last 2 decades in lactating Holstein cows (Pursley et al., 1997;Santos et al., 2017).Helmer and Britt (1986) used 1,000 IU of hCG from d 2 to 4 of the estrous cycle to increase circulating concentrations of P4 during diestrus of dairy heifers.Although hCG treatment increased serum P4 concentrations during mid-diestrus, P/AI was not different between treated and nontreated cows.In the same study, the range of days chosen for the treatment raised questions about ovulatory response to hCG treatment, which was not confirmed by manual palpation or ultrasonography examination.In a study conducted by our research team (Cunha et al., 2021a), hCG administered in early diestrus (d 5-7) reduced the proportion of multiparous Holstein cows detected in estrus by activity monitors during a period of 32 d after the previous estrus.However, hCG did not affect estrus detection rate in lactating Jersey cows and increased P/AI in primiparous Jersey cows.In our subsequent study, hCG was reported to increase the proportion of cows with irregular estrous cycle, which might help to explain reduced proportion of multiparous Holstein cows detected in estrous by activity monitor in our previous study.Regardless of the higher ovulatory response to hCG in TAI programs and despite of its capacity to increase circulating P4 concentrations, to this date research shows that hCG use before insemination has been a double-edged sword, and factors such as breed and parity have to be considered in future studies.

hCG USE AFTER INSEMINATION OR PRIOR TO EMBRYO TRANSFER
Progesterone is a crucial hormone in reproductive events related to the establishment and maintenance of pregnancy in cattle.Circulating P4 concentrations during the early postconception period promotes changes in the uterine environment associated with time or duration of the expression pattern of genes in the glandular endometrium cells that contribute to the composition of histotroph (Forde et al., 2009).Further, systemic P4 concentrations were positively associated with improvements in conceptus elongation, production of IFNτ, and pregnancy rates (Inskeep, 2004;McNeill et al., 2006;Clemente et al., 2009).Rizos et al. (2012) determined that the increase in serum P4 resulted from accessory CL induced by hCG on d 5 of the estrous cycle in beef heifers increased conceptus elongation on d 14 (7 d after embryo transfer).After successful confirmation of accessory CL presence and increased luteal volume induced by hCG, conceptus from treated heifers were longer, wider, and had greater total area than conceptus from control heifers.However, the mean IFNτ production from these embryos in vitro was not significantly different between treatments, even though there was a positive correlation between conceptus length or area and IFNτ production.
Given the importance of systemic P4 concentrations postconception for pregnancy, studies have investigated the effect of hCG on P/AI or pregnancies per embryo transfer (P/ET) (Vasconcelos et al., 2011;Niles et al., 2019).The main objective of those studies was to decrease pregnancy loss by inducing ovulation of the first wave DF of the estrous cycle to form an accessory CL.A study performed by Nascimento et al. (2013) tested the effect of hCG administered to lactating dairy cows on d 5 after AI.The results indicated that hCG improved P/AI in primiparous cows but not in multiparous cows.However, in the same study, a meta-analysis showed that most studies had inconsistent results related to pregnancy outcomes.Besbaci et al. (2020), in another meta-analysis, showed that treatments with GnRH (buserelin, gonadorelin, and lecirelin) or hCG improved P/AI only in cows with low fertility.In contrast, hCG or GnRH treatment of cows with very good fertility did not benefit.In that study, the authors categorized cows according to P/AI in the control group of each study (very poor, P/AI <30%; poor, P/AI 30-45%; good, P/ AI 45.1-60%; and very good, P/AI >60%) and compared fertility outcomes from cows that were treated with hCG or GnRH between 4 and 15 d after AI with controls.The potential causes for such fertility differences between studies could not be explored in-depth, and the main speculation was related to concentrations of P4 after AI.Perhaps cows with good fertility do not need treatment to improve luteal function because they already have enough circulating P4 concentrations for adequate embryo development and subsequent pregnancy signaling.Starbuck et al. (2001) indicated that a low embryo survival rate is associated with both low (<3 ng/mL) and high (>9 ng/mL) circulating P4 concentrations.In another study, Niles et al. (2019) assessed the effect of treatment with hCG on d 7 after AI or at the time of in vitro-fertilized embryo transfer on circulating concentrations of P4 and pregnancy-specific protein B, IFNτ stimulated gene 15, and P/AI or P/ET in Furthermore, previous studies using hCG after AI found an interaction between hCG treatment and parity.In those studies, hCG increased P/AI only in primiparous lactating dairy cows (Nascimento et al., 2013;Zolini et al., 2019;Besbaci et al., 2020).Yet, the reason for an hCG fertility effect only in primiparous cows is still to be determined.It may be related to the many physiological and pathological differences among primiparous and multiparous cows, including lower milk production, smaller body size, reduced incidence of diseases, greater fertility, and increased incidence of anovulation (Bamber et al., 2009;Dubuc et al., 2012).Additionally, Zolini et al. ( 2019) also observed an interaction between hCG treatment 5 d after TAI and genotype at coenzyme Q9 locus on P/AI in lactating dairy cows, indicating that genotype may also play a role in hCG treatment efficiency.

CONCLUSIONS
Previous studies provide compelling evidence that hCG administered during late metestrus or early diestrus effectively increases circulating P4 concentrations during the estrous by inducing the formation of an accessory CL or increasing the steroidogenesis capacity of an original CL in dairy cows and heifers.Although systemic P4 concentrations during the estrous cycle or early embryonic development were positively associated with higher fertility in cattle, the use of hCG before or after AI or embryo transfer as a fertility treatment had inconsistent results.For instance, studies using hCG on d 5 to 7 after AI have shown no significant increase in P/AI or P/AI improvement in only primiparous lactating dairy cows.It seems that parity, breed, and even genotype may also affect the hCG effect on fertility outcomes; however, the mechanisms involved in these interactions are not clear.Recent work indicates an effect of hCG when used from d 5 to 7 of the estrous cycle on estrous cycle length, follicular and luteal dynamics during the estrous cycle, follicular wave number, and lutelosyis in nonpregnant lactating dairy cows.These results indicate that there are still gaps in knowledge in the effects of hCG on reproductive physiology in dairy cattle that need to be elucidated to find applications of hCG as a reliable fertility tool in cattle.
Figure 1.Graphic representation of LH, human chorionic gonadotropin (hCG), and the LH/hCG receptor.Granulosa cells from a dominant follicle express LH/hCG receptors and, due to its similarity with LH, hCG binds to the same receptor.
Cunha and Martins: LITERATURE REVIEW: EFFECTS OF hCG ON THE ESTROUS CYCLE et al. (

Figure 2 .
Figure2.Schematic representation of steps needed for GnRH or human chorionic gonadotropin (hCG) to cause ovulation.After i.m.GnRH administration, GnRH is absorbed and transported to the central nervous system, more specifically to the pituitary gland, releasing LH and FSH.Subsequently, LH is transported via circulation to the ovary, binding to its receptor on granulosa cells of the dominant follicle, causing ovulation.In contrast, after hCG i.m. administration and absorption, hCG acts directly on granulosa cells because of its LH-like activity.
Cunha and Martins: LITERATURE REVIEW: EFFECTS OF hCG ON THE ESTROUS CYCLE nulliparous Holstein heifers.Treatment with hCG was related to increased serum P4 concentrations from d 11 to 67.Although P/AI or P/ET at d 32, serum PSPB concentrations from d 11 to 67 of pregnancy, and relative IFNτ stimulated gene 15 mRNA expression on d 18 or 20 did not differ among treatments, hCG-treated recipient heifers had reduced pregnancy loss between d 32 and 67 of gestation.