Effect of 200 μg of gonadorelin hydrochloride at the first GnRH of a CIDR Synch program on ovulation rate and pregnancies per AI in Holstein heifers

The initial ovulatory response during synchronization programs is often low in dairy heifers, largely due to follicular dynamics and hormonal dynamics. Specifically, the progesterone concentration ( P4 ) at the time of the first GnRH treatment in a breeding program can influence the LH response, often resulting in a suboptimal ovulatory response. The objective of this study was to determine the effect of the highest label dose 200 μg (100 μg vs. 200 μg) of GnRH (50 μg gonadorelin hydrochloride per mL; Factrel ® ; Zoetis Inc. Madison, NJ) at the first GnRH of a 6-d CoSynch plus P4 device program on ovulatory response and pregnancy per AI ( P/AI ) in first service in Holstein heifers. A total of 1308 Holstein heifers were randomly allocated at the beginning of a 6-d CIDR-Synch program, Day 0, to receive either i.m. treatment of 100 μg (2CC, n = 655) or 200 μg (4CC, n = 653) of GnRH. Also, at Day 0, heifers received an intravaginal insert with 1.38 g of P4 (Eazi-Breed CIDR ® Cattle Insert; Zoetis Inc., Madison, NJ). On Day 6, the insert was removed, and i.m. treatment of 25 mg of PGF 2α (12.5 mg dinoprost tromethamine/ mL; Lutalyse ® HighCon Injection Zoetis) was administered. On Day 7, a second i.m. treatment of 25 mg of PGF 2α was given, followed on Day 9 by concurrent i.m. treatment of 100 μg of GnRH and timed AI ( TAI ). A subset of 396 heifers had their ovaries scanned to evaluate ovulatory response, and blood samples were collected to measure the serum concentration of P4 at Day 0 and Day 6 of the study. The P4 concentrations at Day 0 were categorized as Low (≤3ng/mL) or High (>3ng/mL). The ovulatory response was greater for heifers receiving 4CC than 2CC at Day 0 (54.7% vs. 42.8%). The ovulatory response was greater for Low P4 than High P4 at Day 0 (54.3% vs. 37.8%). However, there was not an interaction between treatment and P4 concentrations (Low P4 2CC = 48.6% vs. High P4 2CC = 30.0%; Low P4 4CC = 60.0% vs. High P4 4CC = 45.5%). The ROC curve analysis indicates that P4 concentrations at Day 0 treatment could predict the ovulatory response, although the area under the curve was only 0.6. As expected, heifers that ovulated had increased P/AI (No = 55.6% vs. Yes = 67.7%); however, there was no effect of treatment on P/AI (2CC = 63.3% vs. 4CC = 59.6%), nor interactions between treatment and ovulation and treatment and P4 (HIGH vs LOW) for pregnancy outcomes. In summary, P4 concentration and increasing the dose of GnRH at Day 0 positively impacted ovulatory response in Holstein heifers. However, there was no interaction between treatment and P4 on ovulation and no subsequent impact of GnRH dose on P/AI.


INTRODUCTION
Synchronization of ovulation has been widely recognized as an effective method for optimizing pregnancy outcomes in heifers, with studies supporting its benefits in obtaining high pregnancy rates in a cost-effective manner (Lima et al., 2011, Lima et al., 2013, Silva et al., 2015).However, the initial ovulatory response during synchronization programs in dairy heifers is usually lower (15 to 35%) than in dairy cows (50 to 60%) (Pursley et al., 1995, Sartori et al., 2004, Bello et al., 2006, Rabaglino et al., 2010, Lima et al., 2013, Lauber et al., 2021) often due to variations in follicular dynamics and progesterone (P4) secretion (Sirois and Fortune, 1988, Sartori et al., 2004, Lima et al., 2013).Dairy cows tend to have predominantly 2 follicular waves (Bleach et al., 2004, Sartori et al., 2004, Burns et al., 2005), but there have been studies reporting the existence of 3 follicular waves in more than 40% of the dairy heifers (Sirois and Fortune, 1988, Kulick et al., 2001, Sartori et al., 2004).Heifers exhibiting a pattern of 3 follicular waves experience intervals of approximately 7 d between waves, with the first, second, and third waves initiating around d 2, 9, and 16, respectively.This, in turn, could reduce the window of time for follicles to ovulate when these heifers are treated with GnRH (Savio et al., 1988, Sirois and Fortune, 1988, Sartori et al., 2004).
Another physiological aspect that has been shown to impact GnRH-induced ovulation at the beginning of the estrous synchronization programs is the plasma concentration of P4.Indeed, findings from previous studies demonstrated a higher P4 concentration is consistently associated with lower plasma concentrations of LH in responses to GnRH in beef heifers (Colazo et al., 2008, Batista et al., 2017), dairy heifers (Lima et al., 2013), and dairy cows (Giordano et al., 2012).Hence, the P4 milieu is an integral component of the hormonal landscape that determines the GnRH response, thereby influencing the ovulatory potential of the treated animal.Indeed, the previous study in dairy heifers demonstrated that heifers in the Low Progesterone group had higher LH release from 45 to 120 min after GnRH treatment and higher ovulatory response (48.4% vs. 19.0%)when compared with heifers in High Progesterone group (Lima et al., 2013).
An approach to mitigate the reduced LH release in lactating dairy cows with high progesterone was increasing the dose of GnRH in the estrous synchronization program (Giordano et al., 2012).Interestingly, it was reported that when a double dose of GnRH of the current standard estrous synchronization dose (200 μg vs. 100 μg) was administered, it increased the plasma concentration of LH released (Giordano et al., 2012).The translational potential of increased LH release with a double dose of GnRH for estrous synchronization was further assessed in other studies revealing an improvement in the ovulatory and pregnancy responses in dairy cows (Martinez et al., 2021b, Lima et al., 2022, Valdes-Arciniega et al., 2023).A recent study on dairy heifers demonstrated that those receiving a double dose of GnRH at the beginning of the program exhibited significantly higher ovulatory responses (Colazo et al., 2023).
Consequently, continuous refinement of synchronization protocols and integration of recent advances in reproductive management is crucial for maximizing the effectiveness of synchronization of ovulation in dairy heifers.The low GnRH-induced ovulatory response of dairy heifers at the beginning of estrous synchronization programs, combined with the negative impact of progesterone on LH release and ovulation suggests that doubling the dose of GnRH offers an opportunity to improve pregnancy outcomes.Therefore, we hypothesized that administering a double dose of gonadorelin hydrochloride (200 μg vs. 100 μg) at the first GnRH injection in a 6-d CIDR Synch program would enhance ovulatory response and first service pregnancies per AI in Holstein heifers.The study objectives were to evaluate the impact of a high dose of gonadorelin hydrochloride on ovulation rate and pregnancy outcomes in Holstein heifers following the 6-d CIDR Synch program.

MATERIALS AND METHODS
All experimental procedures carried out in this study received an ethical review by the Institutional Care and Use Committees of the University of California, Davis (Protocol# 22792).

Heifers, Diets, and Housing
A total of 1,308 nulliparous Holstein heifers received their first insemination, with an average age of 15.2 mo (ranging from 14 to 17 mo), were included in the study conducted between February and May 2022, and were housed in a cattle feeder located in Wellington, Nevada.They had unrestricted access to water and twice daily access to a TMR ration formulated to meet or exceed the nutritional requirements of Holstein heifers weighing 360 kg and gaining 0.8 kg/day (NRC, 2001).Throughout the administration of hormonal treatments, insemination, and pregnancy examination, heifers remained in the same pen until the last pregnancy diagnosis.

Experimental Design and Treatments
Day 0 was defined as the enrollment day of heifers in the study.At Day 0, heifers were evaluated and had their body condition score (BCS) assessed using the previously established scoring system for dairy cattle (2004 Elanco Animal Health, Body condition scoring for dairy replacement heifers, adapted from Ferguson et al., 1994).On Day 0, heifers were ranked in ascending order based on their genomic prediction of daughter pregnancy rate (GDPR).Subsequently, at Day 0, they were randomly assigned to receive either (2CC) 100 μg (n = 655) or (4CC) 200 μg (n = 653) of GnRH as gonadorelin hydrochloride (50 μg gonadorelin hydrochloride per mL; Factrel ® ; Zoetis Inc. Madison, NJ).Also, at Day 0, heifers received a CIDR insert with 1.38 g of P4 (Eazi-Breed CIDR ® Cattle Insert; Zoetis Inc., Madison, NJ).The CIDR insert was removed 6 d later on Day 6, and concurrently, heifers received a 25 mg i.m. injection of prostaglandin F2 α (PGF 2α ) as 25 mg of dinoprost tromethamine (12.5 mg dinoprost/ mL; Lutalyse ® HighCon Injection Zoetis Inc., Madison, NJ).Heifers then received, on Day 7, a second dose of PGF 2α 24 h after CIDR removal.Two days later, on Day 9, all heifers received a second dose of 100 μg of GnRH concurrent with timed AI and were performed by 3 technicians with sexed sorted semen: Holstein (n = 999), Crossbred (n = 152), Simental (n = 80) and Jersey (n = 77).(Figure 1; Table 1).

Ultrasonography of Ovaries and Evaluation of Ovulatory Responses
Each week, a cohort of 100 heifers was selected for the study, and from this group, 30 underwent ovarian evaluations and blood sample collections, totaling 396 heifers by the end of the study.These procedures were repeated on Day 6. Ovarian evaluations were performed using the Easi-Scan Go Veterinary Ultrasound Scanner (IMV Imaging, Rochester, MN).Presence of corpus luteum (CL) larger than 14 mm and follicles equal to or greater than 10 mm in diameter were documented.Ovulation at the start of the timed AI (TAI) program was assumed if a heifer had a follicle equal to or greater than 10 mm on Day 0, and a newly formed CL was observed in the same ovary on Day 6. Heifers with follicles smaller than 10 mm on study Day 0 but with a new CL on study Day 6 were considered to have ovulated before study Day 0.

Blood Sampling and Analysis of Progesterone Concentrations
Blood samples were taken from 396 heifers, which were from the same subset previously mentioned in the ultrasonography examination section.Blood was drawn on study Day 0 and Day 6 by puncturing the median coccygeal vein or artery, using evacuated tubes (Becton Dickinson, Franklin Lakes, NJ) containing K2 EDTA for plasma separation.Immediately after collection, samples were placed on ice and refrigerated until transported to the laboratory.Upon arrival at the labora-tory, blood tubes were centrifuged at 2,000 × g for 15 min at 5°C, and a 2 mL plasma aliquot was frozen at −80°C for later analysis.The P4 was analyzed using the competitive enzyme immunoassay of Munro and Stabenfeldt (1984), as previously described in Gomez et al. ( 2018) Swanepoel et al. (2020).The average sensitivity of the assay was 1.17 ng/mL, calculated at 2 standard deviations below the mean counts per minute at maximum binding.All samples were analyzed in duplicate, and the intra-assay coefficient of variation averaged 10.7%.The inter-assay coefficients of variation were 10.9%.Heifers were categorized based on the plasma concentration of P4.Heifers with plasma P4 concentration greater than 3 ng/mL were classified as High, while heifers with plasma P4 concentration lower or equal to 3 ng/mL were classified as Low.The rationale for using the 3.0 ng/ml was 2-fold.First, a previous study in dairy heifers from our group indicated that a concentration of P4 > 3.5 ng/ml at the time at GnRH might compromise LH release and ovulation in dairy heifers (Lima et al., 2013).Furthermore, a study demonstrated that cows with high P4 concentration at the time released more LH when receiving 200 vs. 100 ug of gonadorelin diacetate tetrahydrate (Giordano et al., 2012).

Pregnancy Diagnosis and Calculation of Pregnancy per AI and Pregnancy Loss
Pregnancy was diagnosed at 37 d post-TAI through transrectal ultrasonography of the uterus using a portable ultrasound device with a 7.5-MHz transrectal probe (Easi-Scan; BCF Technologies USA Ltd.LLC, Rochester, MN).The presence of an amniotic vesicle with a visible embryo heartbeat was used as a criterion to de- .Schematic representation of experimental procedures: 1,308 Holstein heifers were enrolled in the study.BCS were recorded, and the heifers were ranked based on their GDPR.On Day 0, the heifers were then randomly assigned to 2 treatments, either a 100 μg dose (2CC = 655) or a 200 μg dose (4CC = 653) of GnRH (Day 0) as part of a CIDR Synch protocol.Additionally, on Day 0, the heifers were administered a CIDR and GnRH (Day 0).On Day 6, the CIDR was removed, and a shot of PGF was administered.On Day 7, a second shot of PGF was given.Finally, on Day 9, the heifers received 100 μg of GnRH and were artificially inseminated.A subset of 396 heifers underwent ovarian scanning, and blood samples were collected to measure the serum concentration of progesterone (P4) at GnRH (Day 0) and 6 d later.
termine pregnancy.Heifers confirmed pregnant on Day 37 underwent another transrectal palpation of uterine contents around 79 d post-AI.The pregnancy rate per AI (P/AI) was determined by dividing the number of pregnant heifers on d 37 or 79 by the total number of AI recipients.Pregnancy loss was diagnosed as heifers being pregnant at 37 d post-AI but then non-pregnant at 79-or 177 d post-TAI.Pregnancy loss between 37 and 79 d and between 37 and 177 d were calculated by dividing by the number of heifers identified as nonpregnant on Day 79 or 177 by heifers diagnosed pregnant at 37 post-TAI, respectively.

Statistical Analyses
Power analyses were performed to calculate sample sizes using G Power 3 (Universität Düsseldorf).Sample sizes were calculated for the study to allow sufficient power to detect a difference of 7 percentage units in P/ AI between treatments [α = 0.05; β (the probability of a type II error) = 0.20; One-tailed test].The expected P/AI for the first AI was combined for heifers receiving a CoSynch plus progesterone device program is 58% based on previous studies (Lima et al., 2013, Lima et al., 2011).Under these assumptions, a minimum of 597 experimental units per treatment were deemed necessary to test our hypotheses.Additional heifers were added to increase the statistical power and reduce the change of type II error.Also, sample sizes were calculated for the study to allow sufficient power to detect a difference of 15 percentage units in ovulation response between treatments [α = 0.05; β (the probability of a type II error) = 0.20; one-tailed test].The expected ovulation for the first AI combined for heifers receiving a CoSynch plus progesterone device program was between 34.4% and 35.4% based on previous studies (Lima et al., 2013, Lima et al., 2011).Under these assumptions, a minimum of 134 experimental units per treatment were deemed necessary to test our hypotheses.Because of potential attrition, additional heifers were added to both treatments.
Categorical data were analyzed by logistic regression using PROC GLIMMIX of SAS version 9.4 (SAS/ STAT; SAS Institute Inc., Cary, NC), fitting a binary distribution Backward stepwise logistic regression models were used, and variables were continuously removed from the models by the Wald statistic criterion when P > 0.10.The Akaike information criterion (AIC) was used to select the final model variables (stepwise elimination approach).The model with the lowest AIC was used.Descriptive statistics were utilized to assess the pre-enrollment equivalence between treatment groups, for continuous variables such as average age, BCS, and GDPR were performed using PROC MEANS.More- to predict ovulation and pregnancy.Additional models were also performed to assess the impact of P4 at Day 0 on pregnancy and ovulation, with models including treatment effects, categorized P4 on d0 (High >3 ng/ mL and Low ≤3 ng/mL), and their interactions.Differences with P ≤ 0.05 were considered significant, and those with 0.05 < P ≤ 0.10 were considered tendencies.

Descriptive Statistics
The mean and distribution by month age for heifers, the mean and distribution BCS, and the mean and distribution GDPR did not differ for heifers enrolled in the 2CC and 4CC treatments (Table 1).For ovarian characteristics on Day 0 of the study, the presence of one (P = 0.14) or multiple (P = 0.82) follicles, the number of follicles (P = 0.59), and the size of the largest follicle (P = 0.23) were not different for heifers in 2CC or 4CC treatments (Table 2).The presence of one (P = 0.65) or multiple CLs (P = 0.86), the number of CLs (P = 0.87), and the size of the largest CL (P = 0.36) were not different at Day 0 for heifers in 2CC or 4CC treatments (Table 2).The concentrations of P4 at Day 0 tended to be lower (P = 0.06) in the 2CC compared with the 4CC treatment (Table 2).

Ovulatory Responses
Heifers receiving 4CC treatment had a greater ovulatory response (P = 0.01) than heifers receiving 2CC (Figure 2A).Only a small fraction of heifers without follicles equal to or larger than 10 mm at the beginning of the study at (Day 0) had new CL at Days 6 (n = 5, 2CC = 2, and 4CC = 3) at Day 6 indicating very few heifers potentially had ovulation occurring before treatment was administered.Heifers in the Low P4 group had greater (P < 0.001) ovulatory response than heifers in the High P4 group (Figure 2B).However, no interactions were observed between treatment and the P4 group (P = 0.26) for ovulatory response (Figure 2C).Moreover, ROC curve analyses were conducted to assess the predictive ability of P4 concentrations during Day 0 for the ovulatory response.The analysis revealed that P4 concentrations could predict ovulation; however, the area under the curve was only 0.6 (Figure 3), indicating a moderate prediction.

Ovarian Responses on Day 6 of the Study
For ovarian responses, a trend was observed (P = 0.09) for the presence of follicles, with fewer heifers in the 4CC treatment tending to have follicles ≥10 mm when compared with the 2CC treatment (Table 3).The presence of multiple follicles (P = 0.79), the number of follicles (P = 0.52), and the size of the largest follicle (P = 0.86) were not different for heifers in the 2CC and 4CC treatments (Table 3).The presence of CL was not different (P = 0.39) between treatments (Table 3).A trend was observed for the presence of multiple CLs (P = 0.07), with more heifers in the 4CC treatment tending to have multiple CLs.Also, the heifers in the 4CC treatment tended to have more CLs (P = 0.07) than heifers in the 2CC treatment.The size of the largest CL (P = 0.36) was not different between treatments (Table 3).The P4 concentrations on Day 6 of the study did not differ between the treatments (Table 3).
For the subset group of heifers evaluated based on ovarian dynamics (P = 0.45; Figure 4A) and P4 (P = 0.75; Figure 4B), there were no differences in P/AI at 37 post-TAI between 2CC and 4CC treatments, and there were no interactions (P = 0.75) between treatment and P4 groups (Figure 4C).
For the subset of heifers with ovulation data, heifers that ovulated had greater (P = 0.01) P/AI at 37 post-TAI than heifers that failed to ovulate (Figure 4D), but no interactions (P = 0.54) between treatment and ovulation were found (Figure 4E).

DISCUSSION
As anticipated, heifers treated with the doubled GnRH dose displayed an increase in the ovulatory response at the first GnRH treatment of the program, and heifers categorized as Low P4 plasma concentrations at Day 0 had higher ovulatory response than heifers in High P4 group.However, contrary to expectations, the improved ovulatory response at the beginning of the program did not translate into improved pregnancy per AI.While the P4 concentration at the beginning of the  program predicted the ovulatory response and heifers that ovulated had higher pregnancy rates, no interactions between ovulation treatment or P4 and treatment were present for pregnancy outcomes at either Day 79 or 177 post-AI.
The increase in ovulatory response of heifers in the 4CC treatment aligned with results from a recent study on dairy heifers that compared the use of a double dose of GnRH at the beginning of a 5-d CO-Synch protocol, reporting an increase from 27.9% to 51.8% in heifers receiving 200 μg when compared with heifers receiving 100 μg (Colazo et al., 2023).The improved ovulatory response is consistent with previous studies conducted in Holstein dairy cows receiving 200μg vs. 100 μg at the beginning of the breeding OvSynch of a double OvSynch program.Ovulatory response increased from 69.4% to 81.2% (Martinez et al., 2021a), and from 65.0% to 81.3%, for cows treated with 200 μg compared with 100 μg (Martinez et al., 2021;Valdez-Arciniega et al., 2020).Previous studies reported a GnRH, dosedependent LH release response in dairy cows (Souza et al., 2009, Giordano et al., 2012), consistent with ovulatory responses observed in the current studies.Thus, the premise that there is a GnRH dose-related response for ovulation was confirmed herein as previously described (Colazo et al., 2023).
As expected, progesterone concentrations on Day 0 influenced ovulatory response such that heifers in the Low P4 group had an increase in ovulatory response, but there was no interaction with treatment.The lack of interaction suggests that the magnitude of response to treatment was not sufficient to influence responses observed in ovulation between progesterone groups differently.An increase in the ovulatory response for heifers in the Low P4 group from 19.0% to 48.4%, when compared with the High P4 group, was similar to results previously reported (Lima et al., 2013).In that study, heifers in the high progesterone group had a mean plasma concentration of progesterone of 7.35 ng/mL, which was slightly higher than the mean concentration observed in the current study (6.57ng/mL), but that is unlikely to be sufficient to explain the differences in the magnitude of ovulatory response (Lima et al., 2013).Previous studies have found differences in the magnitude of the responses to GnRH in high and low P4 environments, consistently demonstrating an increased ovulatory response in animals within the low P4.For instance, beef heifers (Colazo et al., 2008, Dias et al., 2010) and dairy cows showed a notable increase  in ovulatory response for those animals with low P4 (Giordano et al., 2012, Stevenson andPulley, 2016).Past research has suggested that progesterone can reduce the expression of GnRH receptors and thereby pituitary sensitivity to GnRH (Nett et al., 2002, Rispoli and Nett, 2005, Stevenson and Pulley, 2016).Indeed, progesterone concentration was a significant (if only moderate) predictor for ovulation but was insufficient to translate into improved pregnancy outcomes as anticipated, suggesting that other physiological factors play a role in dairy heifers subjected to estrous synchronization programs.
As anticipated, heifers ovulating at the beginning of the synchronization program had increased P/AI, but surprisingly, the benefits of the increased ovulatory response achieved by the double dose of GnRH did not result in significant differences in P/AI at Days 37, 79, and 177 post AI.The positive association between ovulation at the beginning of the synchronization program and pregnancy outcomes is consistent with studies in dairy cows and heifers (Colazo et al., 2008, Giordano et al., 2012, Lima et al., 2013, Bisinotto et al., 2015).Nonetheless, contrary to our hypothesis, the greater ovulatory response induced by the double dose of GnRH failed to improve pregnancy outcomes, contrasting the findings of a similar recent study (Colazo et al., 2023).In that previous study, heifers receiving 200 μg of GnRH had increased P/AI at 30 d post-AI (75.9% vs. 63.1%)when compared with animals receiving 100 μg (Colazo et al., 2023).These disparities in pregnancy outcomes may potentially be attributed to the differences between the protocols used.In the current study, heifers stayed with the progesterone device for 6 d, while in the previous study, they kept the progesterone device for only 5 d.Thus, in the current study, heifers had follicles that were under a longer period of dominance when the P4 device was removed, which could have made them more susceptible to express estrus earlier, as indicated by the previous studies (Colazo et al., 2023, Gobikrushanth et al., 2023).In that study, the researchers revealed that increasing the dose of GnRH from 100 μg to 200 μg reduced the interval from device removal to estrus from 61.0 to 54.3 h in the heifers ovulating after GnRH treatment (Colazo et al., 2023).Therefore, it is possible that in the current study, the additional day available for follicle development increased the percentage of heifers expressing estrus too early and in an asynchronous manner, relative to the insemination time when receiving a higher dose of GnRH, which might have offset the benefits from the increased ovulatory response in heifers receiving 200 μg of GnRH.Another difference was that the increase in ovulatory response in the previous study was larger (From 27.9% to 51.8%) than in the current study, potentially contributing to the effects of pregnancy observed in their study.
Therefore, it is reasonable to surmise that the lack of improved reproductive outcomes in heifers receiving 200 μg of GnRH in the current study might be a result of the increased percentage of heifer expressing estrus sooner after P4 removal (e.g., < 48 h) that led to an asynchronous ovulation relative to semen availability, ultimately offsetting benefits of the improved ovulation induced by higher dose of GnRH.Even though the results support the importance of ovulation in influencing the outcomes of the timed AI program, it is important to acknowledge that pregnancy rates are influenced by broader and more complex factors such as length of follicle dominance and synchrony between estrus expression, ovulation, and semen availability to support successful fertilization.

CONCLUSION
Overall, the findings of this study indicate that increasing the dose of GnRH from 100 μg to 200 μg at Day 0 of the 6-d CIDR synchronization program enhances the ovulatory response in Holstein heifers.However, despite this improvement in ovulation, no effects were observed on pregnancy outcomes.These results have practical implications for reproductive management strategies in dairy heifers, suggesting that increasing the GnRH dose alone may not be sufficient to improve overall pregnancy.Ultimately, comprehending the complexity of factors influencing pregnancy outcomes in heifers is needed, highlighting the necessity to consider multiple variables when evaluating and predicting pregnancy rates.
Figure1.Schematic representation of experimental procedures: 1,308 Holstein heifers were enrolled in the study.BCS were recorded, and the heifers were ranked based on their GDPR.On Day 0, the heifers were then randomly assigned to 2 treatments, either a 100 μg dose (2CC = 655) or a 200 μg dose (4CC = 653) of GnRH (Day 0) as part of a CIDR Synch protocol.Additionally, on Day 0, the heifers were administered a CIDR and GnRH (Day 0).On Day 6, the CIDR was removed, and a shot of PGF was administered.On Day 7, a second shot of PGF was given.Finally, on Day 9, the heifers received 100 μg of GnRH and were artificially inseminated.A subset of 396 heifers underwent ovarian scanning, and blood samples were collected to measure the serum concentration of progesterone (P4) at GnRH (Day 0) and 6 d later.
Melo et al.: GnRH IN DAIRY HEIFERSTable1.Descriptive data representing the number of heifers (n), and the distribution within treatment groups.mean (±SE), and range for genomic prediction of daughter pregnancy rate (GDPR), average milk production in the first 2 tests (M1), and 305-d mature-milk equivalent (ME305) for the lowest (Q1), second (Q2), third (Q3), and highest (Q4) quartiles of primiparous and multiparous cows, and semen breed typeItem Treatments P-value 2CC 4CCover, categorical variables, including age in months categories, BCS categories (Low from ≤2.75, Medium = 3, and ≥ 3.25, and GDPR categories (Q1 = Lowest GDPR and Q4 = Highest GDPR) were analyzed using PROC FREQ to report proportions.The GLIMMIX procedure of SAS was also utilized to compare 2CC and 4CC enrolled heifers.The study's models for P/AI and pregnancy loss included the effects of treatment, sire, AI technician, and their interactions.Continuous data were analyzed using the GLIMMIX procedure of SAS version 9.4 (SAS/ STAT; SAS Institute Inc., Cary, NC), with models fitting a Gaussian distribution.Logistic regression and receiver operating characteristic (ROC) curves were used to generate a threshold of P4

Figure 2 .
Figure 2. Bar graph illustrating the adjusted ovulatory response (A) in heifers in the 2CC and 4CC (100 μg vs. 200 μg) of GnRH (50 μg gonadorelin hydrochloride per mL; Factrel ® ; Zoetis Inc. Madison, NJ) at the first GnRH of a 6-d CoSynch plus P4 device program) treatments, (B) evaluation of ovulatory response when groups were divided into High and Low progesterone concentrations (High >3.0 ng/mL vs. Low ≤3.0 ng/mL) at Day 0 of the study; and (C) comparison between treatment (2CC and 4CC) within each progesterone concentration (Low and High) at Day 0. Analyses were done from the subset group of heifers (n = 392), and the proportion is listed above the bar graph and standard, whereas the number of cows per treatment is within the bar at the bottom.Error bars represent the standard error of the mean.

Figure 3 .
Figure 3. Evaluation of progesterone concentration at Day 0 as a predictor of ovulation at the beginning of the program/first GnRH of CIDR Synch protocol.AUC = Area under curve.
Melo et al.: GnRH IN DAIRY HEIFERS

Figure 4 .
Figure 4. Bar graphs illustrate the proportion of heifer pregnant at Day 37 post-AI in the subset group according to treatment (A), progesterone group, (B), treatment by progesterone (C) ovulation at Day 0 of study (D), and interaction ovulatory response or not at Day 0 within each treatment group.The proportion of pregnant cows is listed on the bar graph at the top, whereas the number of cows per group is at the bottom.Error bars represent the standard error of the mean.

Table 2 .
Melo et al.: GnRH IN DAIRY HEIFERS Ovarian responses and progesterone (P4) concentrations at Day 0 of a CIDR Synch program * CL presence and number are based on ultrasound.

Table 3 .
Follicular ovarian responses and progesterone (P4) concentrations at Day 6 of a CIDR Synch program

Table 4 .
Pregnancy outcomes for heifers in the 2CC and 4CC treatments