Gonadotropin-Releasing Hormone (GnRH) and Its Agonists in Bovine Reproduction II: Diverse Applications during Insemination, Post-Insemination, Pregnancy, and Postpartum Periods

Simple Summary Simple Summary: This review underscores the positive impact of Gonadotropin-releasing hormone (GnRH) and its agonists across several physiological stages, including estrous synchronization, post-insemination, pregnancy, and the postpartum period. It focuses on their role in overcoming reproductive dysfunctions such as repeat breeder cows, early embryonic loss, and cystic ovarian syndrome. The review also highlights their influence on enhancing the productivity of embryo transfer programs. Additionally, it introduces the emerging field of nano-drug delivery systems for GnRH agonists, highlighting potential benefits. The review aims to improve reproductive efficiency and health management in dairy cattle by emphasizing the critical need for further research and development. Abstract The administration of GnRH and its agonists benefits various aspects of bovine reproductive programs, encompassing physiological stages such as estrous synchronization, post-insemination, pregnancy, and the postpartum period. The positive impact of GnRH administration in overcoming challenges like repeat breeder cows, early embryonic loss prevention, and the management of cystic ovarian disease (COD) is thoroughly surveyed. Furthermore, this review focuses on the significance of GnRH administration during the postpartum period, its role in ovulation induction, and how it enhances the productivity of embryo transfer (ET) programs. An emerging feature of this field is introduced, focusing on nano-drug delivery systems for GnRH agonists, and the potential benefits that may arise from such advancements are highlighted. While this review offers valuable insights into various applications of GnRH in bovine reproduction, it emphasizes the crucial need for further research and development in this field to advance reproductive efficiency and health management in dairy cattle.


Introduction
In bovine reproductive programs, GnRH and its agonists play a pivotal role in regulating various aspects of reproduction throughout estrous synchronization, post-insemination, pregnancy, and the postpartum periods (Figure 1).We have recently reviewed the chemical structure of GnRH agonists and their role in the biosynthesis of gonadotropins by the pituitary.This is important for regulating the reproductive process, specifically emphasizing their application in estrous synchronization [1].This review article illustrates the diverse applications of GnRH agonists across different physiological stages.emphasizing their application in estrous synchronization [1].This review article illustrates the diverse applications of GnRH agonists across different physiological stages.Administering GnRH during artificial insemination (AI) is valuable for repeat breeder cows, as it significantly enhances pregnancy maintenance.This effect is more pronounced in repeat-breeding dairy cows than in their first postpartum insemination, leading to increased conception rates and improved fertility [2].The administration of GnRH has a positive impact on preventing early embryonic mortality in cattle.Approximately 25% of bovine embryos face non-viability within the initial three weeks of gestation due to premature luteolysis and a decrease in progesterone (P4) concentration, which can be addressed by GnRH administration [3].
Injecting GnRH agonists during the beginning of the diestrous phase (days 4 to 9) after AI contributes to sustained luteinization of follicles, potentially boosting embryo survival percentages [4,5].The role of GnRH in delaying luteolysis and influencing follicular dynamics contributes positively to increased conception rates [6,7].However, the impact of GnRH agonists during the diestrus (days 11 to 14) after AI varies among studies, influenced by factors such as synchronization programs, heat stress, and environmental conditions [8][9][10].On the other hand, it is noteworthy that administering GnRH around day 30 after AI didn't have a significant effect on maintaining pregnancy [11].
In managing cystic ovarian disease (COD), specifically "follicular cyst", GnRH administration emerges as a preferred treatment [4].Studies highlight its efficacy in inducing follicular dynamic alterations and contributing to the remission of ovarian cysts [12].Combining GnRH agonists with other hormones and modified breeding programs is recommended for improved therapeutic effects [13].
In the critical postpartum period (within 50 to 60 days postpartum) for dairy herds, hormonal treatments, including GnRH, are employed to synchronize reproductive functions and induce ovulation [14].In beef herds, administering GnRH treatment during the postpartum period to induce ovulation of the first postpartum dominant follicle (DF) during the plateau/early declining phase of DF growth enhances ovarian functions.It reduces calving-to-estrous intervals, thereby positively influencing reproductive performance [15].Administering GnRH during artificial insemination (AI) is valuable for repeat breeder cows, as it significantly enhances pregnancy maintenance.This effect is more pronounced in repeat-breeding dairy cows than in their first postpartum insemination, leading to increased conception rates and improved fertility [2].The administration of GnRH has a positive impact on preventing early embryonic mortality in cattle.Approximately 25% of bovine embryos face non-viability within the initial three weeks of gestation due to premature luteolysis and a decrease in progesterone (P 4 ) concentration, which can be addressed by GnRH administration [3].
Injecting GnRH agonists during the beginning of the diestrous phase (days 4 to 9) after AI contributes to sustained luteinization of follicles, potentially boosting embryo survival percentages [4,5].The role of GnRH in delaying luteolysis and influencing follicular dynamics contributes positively to increased conception rates [6,7].However, the impact of GnRH agonists during the diestrus (days 11 to 14) after AI varies among studies, influenced by factors such as synchronization programs, heat stress, and environmental conditions [8][9][10].On the other hand, it is noteworthy that administering GnRH around day 30 after AI didn't have a significant effect on maintaining pregnancy [11].
In managing cystic ovarian disease (COD), specifically "follicular cyst", GnRH administration emerges as a preferred treatment [4].Studies highlight its efficacy in inducing follicular dynamic alterations and contributing to the remission of ovarian cysts [12].Combining GnRH agonists with other hormones and modified breeding programs is recommended for improved therapeutic effects [13].
In the critical postpartum period (within 50 to 60 days postpartum) for dairy herds, hormonal treatments, including GnRH, are employed to synchronize reproductive functions and induce ovulation [14].In beef herds, administering GnRH treatment during the postpartum period to induce ovulation of the first postpartum dominant follicle (DF) during the plateau/early declining phase of DF growth enhances ovarian functions.It reduces calving-to-estrous intervals, thereby positively influencing reproductive performance [15].
In ET programs involving both in vivo and in vitro-derived embryos, there is a focus on enhancing reproductive efficiency.GnRH, human chorionic gonadotropin (hCG), and their analogs have demonstrated positive outcomes, including increased pregnancy rates and reduced embryonic losses [16][17][18][19].
Furthermore, the emerging field of nano-drug delivery systems for GnRH agonists presents a promising avenue, with studies indicating potential benefits, including reduced doses, enhanced bioavailability, and improved fertility outcomes in different farm animals [20,21].However, further research is needed to fully understand its effectiveness, safety, and economic feasibility in dairy cattle.
In summary, this review provides insights into the multifaceted applications of GnRH and its agonists in bovine reproductive practices, highlighting their diverse roles in optimizing conception rates, preventing pregnancy losses, and overcoming reproductive challenges across various stages.

Enhancing Ovulation Response and Conception Rates at the Onset of Estrus and AI Time
Although fixed-time artificial insemination (FTAI) is widely used in dairy reproductive management programs, estrous detection remains essential.Increased physical activity serves as a secondary indicator of estrus in dairy cattle.Recently, electronic systems such as automatic activity monitoring (AAM) [22] and accelerometer systems [23] have been developed to monitor physical activity continuously.This helps to predict the timing of AI in the dairy industry.Variations in the duration of estrus and the timing of AI relative to ovulation can lead to decreased fertility in some cows.However, AI timing based on activity monitoring systems was acceptable for most cows showing increased activity [24].Studies have shown that administering GnRH at the onset of behavioral estrus (OE) positively affects pregnancy rates by increasing ovulation responses or reducing variation in the interval to ovulation [25].Therefore, several studies recommended utilizing GnRH at the estrous phase to elevate P 4 concentrations during the luteal phase of the subsequent estrous cycle.Table 1 presents findings from various studies that support this recommendation.
In a study conducted during the winter season, it was observed that the administration of GnRH and agonists resulted in enhanced pregnancy rates when given at the time of OE, while no beneficial effects were noted when GnRH was administered later in the period of behavioral estrus [26].Moreover, in cows administered GnRH at the OE, the amplitude of the spontaneous luteinizing hormone (LH) pre-ovulatory release was twice as great as typically detected compared to a second, lesser amplitude induction of LH release when administered later in the period of behavioral estrus.Another study conducted during the summer months demonstrated increased pregnancy rates in dairy cows administered GnRH when detecting behavioral estrus [27].Confirming this, cows administered GnRH agonist, dephereline, before AI under heat-stressed conditions exhibited more significant ovulation and conception rates than those administered natural GnRH [28].Moreover, within 3 h after the OE, the administration of GnRH to dairy cows (250 µg) or a GnRH analog (buserelin, 10 µg) led to a greater amplitude of spontaneous LH peaks, fewer delayed ovulations, and higher P 4 concentrations after ovulation [29,30].In dairy cows with high milk production, the use of GnRH markedly improved conception rates.Additionally, injecting cows with a GnRH analog (Gonadorelin, 200 µg) within 5 hours of detecting the OE through the AAM system enhanced conception rates, primarily when inseminations occurred during autumn months, particularly in cows with postpartum health disorders [22].Other studies have administered GnRH at the time of AI and have demonstrated positive impacts on reproductive performance.For instance, utilizing various doses of GnRH at AI has enhanced conception rates in crossbred dairy cows, with no significant difference observed among different GnRH doses [34].The results from the limited number of published reports on GnRH administration during the period of behavioral estrous onset indicate that the administration of GnRH may have a marked beneficial effect on conception rates under inclement climatic heat conditions.Administering GnRH at AI has been found to mitigate the seasonal variation effect by increasing P 4 concentrations and improving conception rates [28] or by reducing the risk of ovulation failure, thereby increasing pregnancy rates and embryonic survival in dairy cows [33,36].Furthermore, in nulliparous dairy heifers, the fertility of sexed semen can be improved to a comparable level by administering GnRH at AI time [37].In the same context, administering GnRH during AI time in beef cattle has also improved fertility parameters.Administrating GnRH at TAI following 7-day P 4 -based protocols has increased fertility in Bos indicus beef cows [40].Similarly, supplementing GnRH treatment at TAI within estradiol (E 2 )-P 4 -based protocol has enhanced fertility parameters in Bos indicus cows.However, it did not impact pregnancy loss rates [41].
Conversely, several studies have shown that administering GnRH at OE and AI time does not improve conception rates or fertility in dairy cows [23,31,32,35].GnRH agonist, gonadorelin, has shown inconsistent effects on P/AI, possibly due to variations in GnRH potency or the timing of administration.Additionally, it is not advisable to administer GnRH during initial services for dairy cattle [31].Also, administering gonadorelin following estrous detection via the accelerometer system has no significant impact on fertility in dairy cows, regardless of standard or heat-stress conditions [23].Furthermore, it is not recommended to use GnRH at the time of AI due to its inability to enhance fertility in dairy cows [32,35].

After Artificial Insemination (AI)
About 25% of cattle embryos are non-viable within the first 3 weeks of the gestational period.The maintenance of pregnancy during these initial stages relies on the continuous secretion of P 4 by a fully functional corpus luteum (CL), and premature luteolysis is considered a primary factor contributing to early embryonic mortality [3].Over the past 25 years, GnRH-based products have been used as a "holding injection" on the day of AI to enhance the proportion of successful pregnancy, particularly in "repeat breeder" cows [4].Repeat breeding is commonly characterized as a syndrome with various potential etiologies, including genetic or acquired defects in ova, sperm, or embryos during the initial developmental stages, infections or inflammatory processes, endocrine dysfunctions, nutritional or management deficits, and other factors associated with early embryonic loss or spontaneous abortion [42,43].Females exhibiting repeated behavioral estrus, with a third or more unsuccessful service, are categorized as "repeat breeders" [2].
The administration of GnRH for repeat-breeding dairy cows has demonstrated beneficial effects, as presented in Table 2. Several studies have indicated that administering GnRH at the time of AI or immediately after AI could positively impact the fertility of repeat-breeder cows [44][45][46][47][48][49].GnRH administration has been associated with improved pregnancy rates by increasing the concentration of serum P 4 starting 6 days after administration [45][46][47].While it has been confirmed that P 4 levels increase 6 days after GnRH administration in repeat breeders, no positive impact on fertility was observed [48].Additionally, it has been reported that GnRH administration may increase P 4 concentrations 2 days earlier in treated cows compared to controls [44].
Furthermore, GnRH administration could increase the diameter of CL and the number of large luteal cells (LLC) within the CL, leading to increased P 4 concentrations and improved fertility [49].Previous studies observed consistent findings when administering GnRH or hCG when the CL was developing in the early luteal phase, resulting in enhanced fertility in repeat-breeding cows [50].Recently, there has been a trend towards maximizing the efficacy of GnRH in treating repeat breeders by administering it along with P 4 , by injection or inserting progesterone-releasing intravaginal devices (PRID) to support early embryonic development while using non-steroidal anti-inflammatory drugs (NSAIDs) to inhibit the synthesis of prostaglandin F2-alpha (PGF 2α ).Studies have found that combined protocols involving GnRH, exogenous P 4 , and tolfenamic acid [51] or meloxicam [52] effectively treat repeat breeders.These protocols support P 4 concentrations and increase conception rates by up to six times compared to the control group.However, further research in this area is still needed.A meta-analysis of 40 experiments evaluating 27 studies revealed that GnRH administration at the time of AI substantially affected conception rates in repeat-breeding dairy cows (22.5%).In contrast, the positive effect of GnRH was only 5.2% units at the first postpartum AI and 8.0% for the effect of GnRH analogs [53].
Several studies have recommended GnRH administration during the diestrous phase as a potential solution to overcome the impact of repeat-breeding problems in dairy cattle.It has been found that GnRH administration during the diestrous phase (5-7 days post AI) can increase the proportion of cows with an additional CL, which enhances embryo survival [54].Similar results were observed when GnRH was administered between the 7th and 14th days after AI in dairy cows [55] and lactating buffaloes [56].Previous research suggested that GnRH administration on day 12 of the previous cycle improves ovulation and conception rates compared to the control group, and it significantly increases P 4 concentrations in pregnant cows on day 22 [57].However, the administration of either GnRH or P 4 (PRID) did not affect the fertility outcomes of repeat breeder cows when used during diestrus (4-18 days post-AI); nevertheless, they increased the P 4 level in treated cows compared to the control [58]

In the Early Luteal Phase (Days 4 to 9 after AI)
During the diestrous phase of the estrous cycle, an anovulatory large DF undergoes atresia due to the estrous cycle stage (i.e., luteal phase) during which development occurs.This initiates the luteolytic process through E 2 produced by the developing follicle.Follicleinduced luteolysis occurs when E 2 affects the endometrium, whose development and functions are modulated by P 4 , leading to the production and secretion of PGF 2α [6,7].The administration of buserelin (a GnRH agonist) at 3-day intervals between the 12th to 48th days after behavioral estrus prolongs the lifespan of the CL due to sustained luteinization of follicles, resulting in the luteolysis of cells or, in the case of induced ovulation, the development of an accessory CL [60].A total of 6 to 8 days after the last buserelin injection on day 48, a fully functional DF is developed, leading to luteolysis and subsequent expression of behavioral estrus [13].Follicular growth is attenuated in the CL-bearing ovary ipsilateral to the pregnant uterine horn.This localized suppression of folliculogenesis and subsequent E 2 production could enhance the anti-luteolytic functions of the developing embryo by suppressing PGF 2α endometrial secretions.Embryonic mortality may occur if the embryo fails to secrete an anti-luteolytic protein, such as the bovine trophoblast protein-1 complex (bTP-1).Improving embryo survival percentages may be achieved by increasing the duration of CL functions, leading to a prolonged period of embryonic development for optimal secretions of bTP-1, inducing an anti-luteolytic effect [61].
Studies summarized in Table 3 demonstrate that administering GnRH analogs during the early luteal phase significantly affects the reproductive performance of dairy cows.Buserelin treatment induced CL-formation between 3 to 6 days after treatment, with large luteal cells, altering CL volume and P4 concentration in cyclic cows compared to acyclic ones.[62].Gonadorelin administration on day 5 post-AI enhanced accessory CL development and boosted the P 4 serum concentrations, suggesting improved luteal function [63,64].Also, GnRH agonist administration via the epidural route on day 7 significantly improved pregnancy rates and overall reproductive performance [65].Similar results were observed when GnRH and hCG were administered intramuscularly during the 5th-7th day post-AI in dairy cows [66] and beef cows [67].This administration significantly increased the ovulatory response, induced the formation of CL, and reduced the number of cows returning to estrus compared to control [66].It has been demonstrated that administering GnRH around the seventh day post-AI aimed at preventing heat stress and excessively high concentrations of P 4 could potentially enhance the LH release profile in dairy cows.This enhancement in the LH release profile might consequently improve P/AI rates [68].
On the contrary, administering GnRH on day 5 or day 15 post-AI did not result in improved fertility outcomes [69].In a recent study, administering GnRH and hCG injections 5 days after AI showed a non-significant improvement in pregnancy rates [70].However, it was noted that GnRH and hCG exhibited beneficial effects by inhibiting the signal pathways of toll-like receptor 4 (TLR-4) and nuclear factor kappa B (NF-κB) while promoting the expression of leukemia inhibitory factor (LIF) and interleukin-1 (IL-1), thereby enhancing uterine receptivity.This inhibition of the immune response in the uterus created a favorable environment for embryo implantation, potentially leading to improved pregnancy rates [70].
These findings collectively highlight the potential of GnRH agonists during the early luteal phase in optimizing reproductive outcomes in dairy cattle, providing valuable insights for enhancing fertility management strategies in the industry.Several studies have investigated the impact of administering a GnRH agonist postinsemination during the luteal phase of the estrous cycle on conception percentages (Table 3).In dairy cows, the administration of 10 µg buserelin between days 11, 12, and 13 post-AI resulted in an enhanced pregnancy rate (72.4%) compared to non-treated cows (60.9%) [76].Additionally, treatment with 10 µg buserelin decreased the percentage of return to service intervals by 22 days compared to non-treated cows, suggesting that the GnRH agonist delayed luteolysis by influencing the dynamics of follicular functions [76].This delay was beneficial for cows at risk of embryonic losses due to luteolysis during the initial stages of gestation [77].Interestingly, even without a viable embryo in the uterine lumen, follicular development was not required for pregnancy maintenance, as demonstrated by successful pregnancies achieved through priming with E 2 followed by progestin replacement after ET into ovariectomized cows [8].In this context, doses of 10.5 µg or 21.0 µg of GnRH were equally effective when administered on day 12 post-AI, leading to increased reproductive performance in dairy cows [72].Additionally, administration of GnRH analog on day 14 significantly improved reproductive parameters and total milk yield compared to the control group [74].
Conversely, some studies reported no beneficial response to administering GnRH agonists during the post-AI period.For instance, there were no significant effects on conception rates in experiments involving synchronization using a GnRH-PGF2α based program with additional administration of buserelin (8 µg) 12 days after insemination [9,10].Similarly, using a GnRH agonist on days 12 to 14 after AI did not enhance the reproductive performance of dairy cows [71,73].
It was hypothesized that estrous synchronization treatment regimens using PGF 2α might lead to luteal phases of shorter duration, potentially reducing fertility [78].Similar results were observed in lactating cows during the summer when GnRH agonist was administered post-AI, showing no significant impact on conception rates [10].In this case, the lack of effect may be attributed to heat stress causing early embryonic mortality, potentially counteracting any beneficial effects of the GnRH agonist treatment on embryo survival.Therefore, when evaluating fertility responses, it is crucial to consider environmental conditions and reproductive management practices applied when implementing hormonal treatment regimens for FTAI.

During Early Pregnancy Diagnosis (around Day 30 after AI)
In contrast to previous findings [79], GnRH administration around 30 days post-AI (early pregnancy diagnosis period) does not impact embryonic loss during the gestational period, particularly in animals with a single fetus (Table 3).However, in cases of unilateral twin pregnancies, GnRH administration results in more remarkable survival of the two fetuses and an increased percentage of pregnancy in cows with two-fetus pregnancies.These results align with another study [11] where GnRH was administered between days 28 and 34 after insemination.Additionally, administering GnRH injection between days 26 and 71 after insemination did not affect the percentage of pregnancy loss [80].The same results were found after administration of GnRH on day 21 following AI, before pregnancy detection, and it was also demonstrated that GnRH does not alter the reproductive performance in dairy cows between 21 and 42 days post-AI [75].
Similarly, the administration of GnRH via a deslorelin implant on day 27 after insemination did not affect the percentage of cows pregnant.However, there was a lower incidence of pregnancy loss between days 45 and 90 after insemination when accessory CL was induced with this treatment [81].Doubling the GnRH dose at the time of pregnancy diagnosis did not affect pregnancy percentage compared to the administration of a single dose of GnRH [11].

GnRH and Cystic Ovarian Disease (COD) "Follicular Cyst"
A cystic follicle is a prevalent reproductive dysfunction in dairy herds, particularly in cows producing large quantities of milk during the early postpartum period [82].This condition occurs when a large ovarian follicle persists in the functional state in the ovary for an extended period without a CL.It is defined as an anovulatory follicle in the ovary for 7 to 10 days with a diameter ranging from 1.7 to 2.5 cm or larger [83].Terms such as ovarian cyst (OC), cystic ovarian degeneration, cystic ovaries, nymphomania, and virilism are used interchangeably to refer to this dysfunction [38].The interaction between genetic, phenotypic factors, and environmental stressors may lead to a lack of ovulation by affecting the hypothalamic-pituitary axis and inhibiting the secretion of GnRH and/or gonadotropins, resulting in follicular cyst syndrome [4].The presence of follicular cysts may lead to economic losses, including prolonged calving intervals, lower pregnancy rates, decreased net calf crop percentages, and higher herd culling percentages [84].
Due to its small molecular weight, GnRH administration is the preferred treatment for cows with a cystic ovarian follicle.This results in a lack of immune reactions compared to multiple treatments with hCG and LH from other species [85].GnRH and its analogs have been effectively utilized to treat cystic follicular syndrome [12].The first report of GnRH treatment for cows with a follicular cyst indicated 70% to 95% effectiveness 18 to 25 days after treatment [86].GnRH administration is routinely used to induce follicular dynamic alterations in cows with cystic follicles [13].In response to GnRH treatment, LH secretion is stimulated, reaching a maximum concentration within 90 to 150 min after treatment.This leads to luteinizing the follicular cysts and the consequent expression of behavioral estrus within 4 weeks post-treatment.Although ovulation from the cystic follicle may not occur in response to GnRH, there may be ovulation from other follicles at treatment time [82].
Several studies recommend using GnRH or GnRH agonists for the treatment of ovarian cysts (Table 4).It has been found that a single intramuscular injection of buserelin at a dose of 10 or higher is as effective as 10,000 IU hCG, making it a recommended treatment for ovarian follicular cysts in cows [87].Additionally, another study found that the administration of GnRH decreased the prevalence of cystic ovarian follicles by day 7 and day 21 compared to the control group [88].There has also been a recommendation to treat cows with 21 µg of buserelin intramuscularly for beneficial therapeutic effects in dairy cows [89].The administration of lecirelin via epidural administration has shown a beneficial impact on reproductive parameters in treating follicular cysts [90].The intramuscular administration of lecirelin (0.1 mg) has been reported as an effective therapy for ovarian cysts in dairy cows that are over 90 days postpartum following confirmation of the presence of a cystic follicle (>20 mm in diameter) [91].In a comparative study [92], a single administration of buserelin at a concentration of 20 µg had an equal therapeutic effect in cows with ovarian cysts compared to a single injection of 200 µg of fertirelin, attributed to the higher potency of buserelin in inducing the release of LH and follicle-stimulating hormone (FSH) from the pituitary and luteinizing the follicular cyst.Conversely, there were no detectable effects on the values for fertility variables as a result of administering two doses of buserelin and gonadorelin (GnRH agonists) to cystic cows in postpartum periods [93].
Combining GnRH agonists with other hormones, such as PGF 2α and P 4 (P 4 intravaginal device, CIDR) for altering the follicular dynamic results in a shorter period of OC effects on reproductive functions [13].The recommendation for administering PGF 2α at 7 to 10-day intervals after GnRH administration has shown beneficial therapeutic effects in cyst remission, significantly improving values for reproductive variables, including intervals of return to estrus, increased ovulation, and pregnancy percentages [43].Implementing the ovsynch treatment regimen has also been a beneficial therapy protocol for COD in dairy herds [94].Modifying breeding programs, such as imposing the ovsynch treatment regimen for OC syndrome, may lead to greater reproductive performance [95].

Postpartum Ovulation Induction
The interval from parturition to the resumption of ovarian activity is essential in the reproductive cycle of dairy cows, marked by distinct physiological changes and significant commercial implications.Several factors contribute to the occurrence and persistence of anovulatory periods during the postpartum phase, including diminished LH release in late gestation, nutritional status during the peripartum period, mainly if there is a negative energy balance, and the influence of suckling stimulus in lactating cows nursing calves [14].
Ensuring the complete involution and infection-free status of the uterus and the timely resumption of estrous cycles (within 50 to 60 days postpartum) are critical aspects of managing fertility and optimizing reproductive performance in dairy herds [74].Monitoring reproductive processes is particularly vital in seasonal calving systems, where the window for calving and achieving pregnancy is time limited.The aim is to ensure that each cow produces a calf annually [100].
Hormonal treatment plays a significant role in synchronizing reproductive functions and inducing ovulation during the early postpartum period.The presence of follicular wave patterns and follicles larger than 9 mm in diameter in the ovaries prompts the administration of GnRH.This induces an LH surge, leading to ovulation from the DF during the early postpartum period in dairy cattle [14].The subsequent reduction in E 2 concentrations, ovulation triggered by GnRH treatment, and the rise in P 4 concentrations from CL development contribute to improved reproductive outcomes [62].Administering GnRH during the early postpartum period (between days 10 and 14) proves beneficial in enhancing the reproductive performance of cows that have experienced an abnormal puerperium [101].While administering this treatment during the early postpartum period (within days 11 to 25) successfully reduces the postpartum to the estrous interval in cows, it does not significantly impact their subsequent reproductive efficiency [102].Multiple studies involving dairy and beef cattle have consistently shown that using GnRH alone or followed by PGF 2α administration can enhance behavioral estrous responses and pregnancy rates.Significantly, these studies indicate a reduction in embryonic/fetal losses when these treatment regimens are applied [103][104][105].
GnRH administration has improved reproductive efficiency by reducing the calving-toconception period and the number of inseminations per conception [106].GnRH treatment during the postpartum period improves ovarian functions early in the postpartum period, leading to a shorter calving-to-estrous interval, approximately 44.6 ± 9.2 days in GnRHtreated cows compared to untreated ones (110.4 ± 50.4) [107].Single administrations of GnRH analogs between days 21 and 31 postpartum induce LH surges and ovulation, showcasing their potential to improve reproductive outcomes [108].Administering GnRH or hCG to anovulatory anestrous cows nine days before mating increases ovulation rates and may lead to higher pregnancy rates after the first insemination than untreated cows.However, it may not significantly impact overall reproductive performance [109] and the use of GnRH or hCG treatment did not show significant effects on overall reproductive performance.Therefore, the authors proposed the possibility of a positive impact on reproductive performance if these treatments are administered earlier [109].Additionally, administering two doses of GnRH at a 10-day interval may induce ovulation and lead to an estrous cycle [110].
Sub-optimal fertility may result from inseminating at the first estrus postpartum due to the formation of abnormal CL.Nevertheless, a short-term treatment with a P 4 implant may stimulate follicular maturation.Similarly, GnRH treatments may positively influence follicular maturation by increasing P 4 concentrations in anestrous cows.The induction of typical estrous cycles with favorable fertility outcomes in anestrous cows can be achieved by injecting PGF 2α 6 days after GnRH treatment, reducing P 4 concentration within 24 h [68].Therefore, in both estrous cyclic and acyclic cows, implementing GnRH-PGF 2α treatment regimens can synchronize ovarian functions, leading to average fertility outcomes [111].Moreover, ovulation induction in acyclic cows treated with GnRH 7 days before administering PGF 2α (known as Select-Synch) showed 38% and 49% ovulation rates in two experiments, respectively [112].In fixed-time embryo transfer (FTET), the transfer of embryos from donor females to recipient females is scheduled at a predetermined time, usually synchronized with the estrous cycle of the recipients.

In Vivo and In Vitro-Derived Embryos for Transfer into Recipient Cows
Embryo transfer (ET) is an essential biotechnology in animal production employed to breed animals with superior genetics.Despite the increasing application of ET in cattle, conclusive results regarding factors affecting its efficacy remain elusive [113].One significant factor that can impact outcomes is the accurate detection of estrus.To overcome this limitation, breeding protocols designed originally for FTAI, which synchronize the time of ovulation among females, can be adapted for FTET applications in recipient cows [114].
Additionally, the functionality of the CL plays a vital role in maintaining pregnancy post-ET.The concentration of serum P 4 is correlated with CL functions, and P 4 significantly affects conception percentages and in-utero embryo development [113,115].P 4 modifies gene expression for uterine-derived proteins, facilitating embryo development and bTP-1 production.A higher P 4 concentration post-AI is essential for sustaining pregnancy and promoting optimal conceptus growth [116][117][118].To address luteal insufficiency during ET, several hormonal therapies, such as exogenous P 4 , hCG, or GnRH/GnRH analogs, have been identified as potential solutions.
Previous studies reported varied outcomes regarding the effects of exogenous P 4 on pregnancy percentages following ET.While some studies suggest a modest positive effect [119,120], others indicate higher embryonic loss and lower pregnancy percentages with P 4 supplementation four days before ET [121].
Furthermore, numerous studies confirm that administering hCG during ET processes leads to desirable outcomes.Treatment with hCG at the time of ET results in the development of accessory CL, increased P 4 concentrations, and higher pregnancy rates [16,17].Additionally, early-stage embryonic losses are reduced after ET.Treatment with hCG following a P 4 -based treatment regimen for estrous synchronization increases pregnancy percentages significantly in lactating cows receiving in vitro-derived embryos by up to 55% [18].Similar benefits are observed in beef cattle, where hCG treatment during ET regimens results in the development of CL and a higher Doppler score associated with an increased pregnancy percentage [19].
Moreover, the administration of GnRH analogs has been suggested to improve outcomes in ET.GnRH during the diestrous phase induces the development of an accessory CL by either ovulation or luteinization of the DF in recipient cows' ovaries [122].A single injection of GnRH on day 5 of the estrous cycle induces ovulation and accessory CL development, resulting in higher P 4 concentrations and increased pregnancy percentages in heifers receiving in vitro-derived blastocysts [123].Administering GnRH analogs during ET procedures in cattle increases pregnancy percentages at both days 30 and 60 (45.8% and 43% compared with 40% and 37% in the control group, respectively), with a reduction in pregnancy loss (4% compared with 7% in the control group) [124].Similarly, the pregnancy rate per FTET is markedly enhanced by up to 55% at day 35 of gestation by treating recipient females with GnRH analogs on the day of ET [125].Beneficial effects of GnRH on pregnancy percentage were observed when administered on day 11 of gestation with ET [126], while contrasting findings emerged for administration on days 11 and 14 [127,128].A recommendation has been made for combining a single-dose GnRH injection and a P 4 implant to mitigate embryonic losses in cows receiving cryopreserved embryos into recipient females [119].

GnRH Nano-Drug Delivery System
Using nano-drug delivery systems presents a promising approach designed to extend the half-life of bioactive components, facilitate their transport through biological barriers, and ensure sustained delivery to target organs.This innovative method can potentially enhance cellular uptake of bioactive components, allowing for smaller doses than conventional administration, benefiting substances such as hormones, peptides, and drugs [129,130].
While studies have explored the impact of nano-GnRH agonists on reproductive performance and fertility in various farm animals, research on their effects in dairy cattle is currently limited.In rabbits, Hassanein et al. [20] utilized a GnRH agonist (buserelin) loaded on chitosan nanoparticles to induce ovulation, successfully reducing the conventional dose by half without compromising fertility.Similarly, in rabbits, applying chitosan-dextran sulfate GnRH (buserelin) nanoparticles intravaginally with seminal doses for ovulation induction reduced the conventional GnRH agonist dose without compromising reproductive performance variables [21].During the low-breeding season in buffalo cows, where reproduction is typically reduced, the administration of nano-GnRH analog (ovurelin) as a modified ovsynch treatment regimen led to a 50% reduction in the conventional dose, with beneficial effects occurring on fertility and ovarian activity in anestrous cows [131].Applying the same methodology in goats, developing a nano-GnRH analog (gonadorelin) allowed for a reduction of hormonal dose by up to 75%, maintaining fertility and prolificacy without adverse effects [132].Additionally, administering nano-GnRH agonist (ovurelin) as part of the ovsynch treatment regimen proved efficacious in enhancing ovarian activity, blood flow, steroid synthesis (E 2 and P 4 ), and CL function during estrous synchronization in goats [133].
Despite some positive benefits of nano-GnRH treatments on reproductive performance in farm animals, further research is necessary to comprehensively understand their effectiveness, safety, and optimal dosing regimens.Additionally, economic cost and feasibility considerations for producing and administering nanoparticles must be considered.

Conclusions
In conclusion, GnRH and its agonists have important and diverse functions in optimizing bovine reproductive practices.GnRH exhibits diverse applications, from preventing early embryonic mortality to managing cystic ovarian disease and enhancing reproductive efficiency.This review article discusses the functions of hypothalamic GnRH in reproductive processes and various applications of GnRH, such as preventing embryonic mortality, regulating ovarian follicular wave dynamics, inducing ovulation, managing cystic ovarian disease, and utilizing GnRH as a nano-formulation for enhancing reproductive efficiency.Further research and development in this field will contribute to advancing reproductive efficiency and health management in farm animals.

Figure 1 .
Figure 1.The diagram illustrates the various applications of Gonadotropin-releasing hormone (GnRH) and its agonists in bovine reproductive practices.

Figure 1 .
Figure 1.The diagram illustrates the various applications of Gonadotropin-releasing hormone (GnRH) and its agonists in bovine reproductive practices.

Table 1 .
The application of GnRH analogs at the onset of estrus and AI time for improving ovulation response and conception rates in Holstein dairy cows.

Table 2 .
Application of GnRH analogs for treating repeat breeder (RB) dairy cows.

Table 3 .
Administration GnRH analogs post-AI for improving fertility in dairy cattle.

Table 4 .
Therapeutic effect of GnRH injection on cystic ovarian disease (COD) in dairy cattle.