Main Research Findings

1) Administration of the gonadorelin formulation, cystorelin, was shown to result in a larger release in LH, as well as a higher ovulatory rate in Holstein cows.

2) High doses of gonadorelin was found to improve ovulatory responses as well as improve the number of viable pregnancies following artificial insemination. `

Selected Data
1) This research team of Martinez et al conducted a series of three experiments for the purpose of investigating the endocrine and ovarian responses of cattle to different commercial formulations of GnRH. The study focused on evaluating LH, FSH, progesterone, and estradiol concentrations in serum or plasma after GnRH administration, as well as the effects on ovulation and follicular dynamics [1].
The first experiment was a preliminary study conducted on eight nonlactating Holstein cows of unknown estrous stages. These animals were housed at the Western College of Veterinary Medicine, University of Saskatchewan, and were housed with unrestricted access to grass-alfalfa hay and water. The cows were randomly assigned to receive an intramuscular injection of 100 μg gonadorelin diacetate tetrahydrate in one of two formulations: either 2 mL of Cystorelin (C) or 1 mL of Fertagyl (FE). Blood samples were collected via jugular venipuncture at 0, 1, 2, and 4 hours after treatment to evaluate LH concentrations in the serum.
The second experiment involved 50 nonlactating Holstein cows weighing between 386 and 489 kg. These cows were housed similarly to those in experiment 1 and provided ad libitum access to hay, water, and cobalt-iodized salt. Luteolysis was induced in all cows using a 500 μg IM dose of cloprostenol. Ovulation was monitored daily using transrectal ultrasonography and defined as day 0. On day 6 or 7 post-ovulation, cows were randomly allocated to one of three treatment groups with 10 cows included in each, and were designated to receive one of the following intramuscularly injected GnRH treatments, including: 100 μg gonadorelin diacetate tetrahydrate in 2 mL of C, 1 mL of FE, or 100 μg gonadorelin hydrochloride in 2 mL of Factrel (FA) [1].
Blood was drawn at 0, 1, 2, 4, and 6 hours after GnRH administration to assess plasma levels of estradiol, progesterone, LH, and FSH. Ovarian examinations were conducted daily from days 0 to 6 and then twice daily until ovulation or up to day 12. A replication of the experiment was carried out with 10 cows each receiving either C or FE, with blood samples collected at 0, 1, 2, 3, and 4 hours for hormonal analysis and ultrasonography performed in the same manner as in the initial phase [1].
In the third experiment, 30 cross-bred beef heifers, weighing between 350 and 400 kg, were housed outdoors in feedlot pens at the Goodale Research Farm, University of Saskatchewan. These heifers were fed barley silage and had free access to water. Luteolysis was again induced with a 500 μg intramuscular dose of cloprostenol, similar to experiment 2. On days 6 or 7 after ovulation, heifers were randomly assigned to receive one of the three GnRH treatments used in experiment 2. Blood samples were collected at 0, 0.5, 1, 1.5, 2, and 4 hours after treatment for measurement of plasma estradiol, progesterone, and LH. Ovarian assessments were conducted similarly to experiment 2 [1].
Blood samples collected for hormonal analysis were placed in heparinized tubes, kept at 4°C, and centrifuged within 4 hours; plasma was then frozen until analyzed. LH concentrations were determined using a double-antibody radioimmunoassay and expressed in terms of NIDDK-bLH4. The assay had a sensitivity of 0.06 ng/mL, with intra- and interassay coefficients of variation of 16.9% and 4.8%, respectively. Peak LH concentration was defined as the highest mean concentration observed after GnRH treatment.
FSH concentrations were measured via a liquid-phase antibody radioimmunoassay, using NIDDK anti-oFSH-1 as the first antibody and USDA-bFSH-1 for standardization. The assay had a sensitivity of 0.125 ng/mL, with intra- and interassay variation of 10.7% and 6.5%, respectively. Progesterone was extracted using hexane from 200 μL plasma samples, and the radioimmunoassay had a sensitivity of 0.1 ng/mL. Intra- and interassay coefficients of variation were 12.0% and 13.8%, based on average concentrations of 1.36 and 0.44 ng/mL, respectively. Estradiol was extracted with ethyl ether and measured with a sensitivity of 0.5 pg/mL. The intra-assay and interassay variations were 11.7% and 10.2%, with mean concentrations of 11.6 and 23.2 pg/mL [1].
Overall, this multi-phase study evaluated the effects of different GnRH formulations on hormonal dynamics and ovarian responses in both dairy cows and beef heifers. By assessing hormone concentrations, ovulation timing, and follicular wave patterns, the researchers aimed to determine the relative efficacy and endocrine profiles associated with each commercial GnRH product [1].
2) The research team of Valdés-Arciniega et al conducted a large-scale study in order to evaluate the effects of two different doses of GnRH on ovulatory and reproductive outcomes in lactating Holstein cows. The experiment took place on a commercial dairy farm with approximately 2,450 lactating cows. Cows were managed in a cross-ventilated barn with separate pens for primiparous (first-lactation) and multiparous (multiple-lactation) cows. All animals had continuous access to water and were fed a total mixed ration twice daily. The ration primarily consisted of corn silage and haylage and was formulated to meet or exceed nutritional recommendations for lactating cows. Pens were equipped with headlocks and freestalls, which were bedded with recycled sand cleaned and replenished every four days [2].
Cows were milked three times a day, with milk yield recorded at each session using electronic flow meters. Data was stored in Dairy Comp 305 software. During the experiment, the average daily milk yield was 42.6 kg per cow, with a herd rolling average of 14,215.6 kg and average milk fat and protein percentages of 4.16% and 3.22%, respectively. A total of 1,909 lactating Holstein cows were enrolled, including 719 primiparous and 1,191 multiparous cows.
Treatments were administered by trained personnel using standard single-dose syringes and 18-gauge needles injected into the semimembranosus or semitendinosus muscles. Hormonal treatments included gonadorelin hydrochloride and dinoprost tromethamine. Genomic testing was conducted on 1,835 of the cows providing genomic predictions for traits like daughter pregnancy rate (GDPR), cow conception rate (GCCR), and the Dairy Wellness Profit Index (DWP) [2].
Cows were assigned to receive either a low dose of 100 μg GnRH or a high dose of 200 μg GnRH at the first GnRH treatment (G1). Assignment was randomized based on odd or even ear tag numbers. The administration protocol consisted of the following sequence: gonadorelin hydrochloride (G1) → 7 days later, dinoprost tromethamine (PG1) → 24 hours later, second dinoprost tromethamine → ~32 hours later, second gonadorelin hydrochloride (G2) → ~16 hours later, timed artificial insemination (TAI).
Primiparous cows received G1 between 69 and 75 days in milk (DIM), while multiparous cows received G1 between 59 and 65 DIM. In the final two months of the study, the G1 timing for both groups was standardized to 64–75 DIM. All subsequent gonadorelin hydrochloride doses in the protocol used the 100 μg dose, and all dinoprost tromethamine injections were 25 mg [2].
Tail paint was applied to identify cows showing estrus around the time of G2. Four artificial insemination (AI) technicians, blinded to the treatment groups, performed inseminations. Cows that showed estrus on the day of G2 were inseminated both that day and the next. Some cows were not inseminated and were instead used as recipients for the farm’s embryo transfer program.
Ovarian ultrasonography was performed at two critical points: before G1 and G2 and again 40 to 48 hours later to assess ovulatory response. In total, 1,293 cows were examined at G1 and 1,019 at G2. Ovarian scans were performed using a real-time B-mode portable ultrasound device with a 7.5-MHz linear transducer. Videos were recorded and stored using specialized ultrasound software [2].
Ovulation was identified by the disappearance of one or more follicles measuring ≥9 mm and the appearance of a new corpus luteum (CL) within 48 hours of GnRH treatment. Follicular and luteal structures were measured by capturing measurements for height and width and calculating the mean diameter. If a CL had a cavity, its area was subtracted from the total. Total luteal area was determined by summing the CL areas from both ovaries [2].
Progesterone concentrations were measured using a commercial radioimmunoassay kit validated for bovine serum. Quality control samples at high concentrations of 4.0 ng/mL and low concentrations of 1.5 ng/mL, were run in triplicate throughout each assay. The average assay sensitivity was 0.04 ng/mL, with intra- and interassay coefficients of variation at 7.3% and 6.1%, respectively.
This comprehensive, randomized field study was designed to assess the effects of different GnRH dosages on reproductive performance and hormonal profiles in lactating Holstein cows using synchronization protocol. Cows were maintained under typical commercial dairy conditions, and reproductive management included AI, detailed ultrasonographic evaluation, and precise hormonal monitoring. By integrating genomic data, reproductive performance, and physiological responses, this study aimed to inform optimized fertility strategies for modern dairy production [2].

Discussion
1) This multi-phase experimental study conducted by the research team of Martinez et al reports on the hormonal and follicular responses observed in three experiments designed to compare different commercial GnRH products in cattle. The studies evaluated LH, FSH, estradiol, and progesterone responses, as well as ovulatory outcomes and follicular wave dynamics in dairy cows and beef heifers following GnRH administration [1].
In the first experiment, the effect of time on serum LH concentrations following GnRH treatment was statistically significant, with levels increasing at 1 or 2 hours post-treatment and returning to baseline by 4 hours. However, there was no significant effect of treatment or the interaction between treatment and time. Cows treated with the C formulation tended to have higher mean LH concentrations than those treated with an average dose of 2.0 ± 0.4 ng/mL of the FE formulation, although this difference did not reach statistical significance [1].

Figure 1: Changes in plasma LH concentrations over the four hours following treatment with either C of FE during experiment 1.
The second experiment was conducted in two phases, each assessing plasma hormone levels and follicular outcomes following administration of one of three GnRH formulations including: C, FE, or FA. Results reported from phase 1 revealed that baseline estradiol and progesterone levels did not differ significantly among the three treatment groups. There was a strong effect of time on plasma LH concentrations, along with a near-significant treatment effect and a trend toward a treatment-by-time interaction. Peak LH concentrations were significantly higher in the C group with an average of 6.6 ± 1.4 ng/mL than in the FE and FA groups with averages of 4.7 ± 0.8 ng/mL and 3.8 ± 0.5 ng/mL, respectively, with no significant difference between FE and FA. In all groups, 50% of cows peaked at 1 hour and 50% at 2 hours post-treatment [1].
Plasma FSH levels showed a significant effect of time, but not of treatment or treatment-by-time interaction. FSH peaked at 1 hour in the FE group and at 2 hours in both the C and FA groups. Several animals were excluded from follicular analysis due to small dominant follicle size of <9 mm, or lack of new follicular wave emergence. Among the remaining cows, ovulatory rate tended to be higher in the C group at 100% compared to the FE and FA groups, at 56% and 57%, respectively, although this was not deemed statistically significant. The average day of emergence of the next follicular wave was similar across treatments, with mean values of 1.7, 1.9, and 2.3 days for C, FE, and FA groups, respectively.
Results reported from the second phase of the experiment found that baseline estradiol and progesterone concentrations were not significantly different between the C and FE groups. However, plasma LH concentrations showed significant effects of treatment, time, and treatment-by-time interaction. Mean LH concentrations were higher in the C group at 4.1 ± 0.4 ng/mL, than in the FE group at 2.3 ± 0.4 ng/mL, with the largest difference occurring 2 hours post-treatment. Peak LH occurred at 2 hours in the C group at 7.9 ± 1.2 ng/mL and at 1 hour in the FE group at 4.2 ± 0.5 ng/mL, with 40% of C-treated cows peaking at 1 hour and 60% at 2 hours; the reverse pattern was seen in the FE group [1].

Figure 2: Changes in plasma LH concentrations over the four hours following treatment with either C of FE during experiment 2.
Plasma FSH concentrations showed a significant effect of time, but not of treatment or the interaction between treatment and time. All cows had dominant follicles of at least 14 mm at the time of treatment, with no differences between groups. Ovulatory response tended to be higher in the C group at 90% than in the FE group at 60%, although this did not reach statistical significance. The intervals from treatment to ovulation were approximately 36–37 hours and to follicular wave emergence was 1.8 vs. 2.2 days for C and FE, respectively. These values did not differ significantly, though the latter showed a trend. Since no significant phase effects were detected on any follicular endpoint, data from both phases were combined, including comparisons of dominant follicle diameter at treatment, interval to ovulation, and time to new follicular wave emergence [1].
Results of the third experiment reported that estradiol and progesterone levels at the time of treatment did not differ among the C, FE, and FA groups. However, there were significant effects of treatment and time, and a near-significant treatment-by-time interaction on plasma LH concentrations. LH levels peaked at 1 hour post-treatment in all groups, but were significantly higher in the C group than in the FE and FA groups, which did not differ from each other. The greatest difference in LH occurred 1.5 hours after treatment.

Figure 3: Changes in plasma LH concentrations over the four hours following treatment with either C of FE during experiment 3.
All heifers had dominant follicles ≥10 mm at the time of treatment, and treatment did not affect any follicular endpoints. While there was no difference among treatment groups on the day of follicular wave emergence, ovulation status significantly influenced the timing of new wave emergence. Heifers that ovulated had earlier and less variable follicular wave emergence with a mean of 1.5 ± 0.1 days, compared to non-ovulators with a mean of 2.7 ± 1.3 days [1].
Across all three experiments, GnRH treatment consistently induced an LH surge peaking around 1–2 hours post-administration and returning to baseline by 4 hours. C treatment generally elicited higher LH concentrations and higher ovulation rates compared to treatment with FE and FA. FSH responses were less affected by treatment and more influenced by time. Follicular responses, particularly the timing of new wave emergence, were influenced by ovulatory status rather than treatment per se. Collectively, these results suggest that while all GnRH products effectively stimulate LH release and support ovulation, Cystorelin may induce a more robust LH surge, potentially leading to improved ovulatory outcomes in both dairy cows and beef heifers [1].
2) This study conducted by researchers Valdés-Arciniega et al evaluated the effects of administering two different doses of gonadorelin hydrochloride, 100 μg vs. 200 μg, during the first GnRH treatment in a TAI protocol in lactating Holstein cows. The study aimed to assess how dosage, parity, progesterone levels, and ovulatory response influence ovarian physiology, pregnancy outcomes, and the relationship with genomic fertility traits [2].
Initial descriptive statistics showed no significant differences between treatment groups in GDPR, GCCR, milk production, or baseline ovarian parameters at G1. However, parity had notable effects as primiparous cows produced less milk and had higher circulating progesterone concentrations and a lower proportion of multiple CL than multiparous cows, likely due to lower dry matter intake and thus less hepatic clearance of progesterone.
As hypothesized, cows receiving 200 μg of GnRH had significantly higher ovulatory response to G1 at 81.3%, compared with those receiving 100 μg that had an ovulatory response of 65.1%, regardless of parity. Ovulatory response did not differ between primiparous and multiparous cows, and no treatment and parity interaction was observed. Larger doses of GnRH enhanced ovulatory response across all serum progesterone tertiles, especially mitigating the suppressive effects of high circulating progesterone, which typically reduce the LH surge. This aligns with prior findings that elevated progesterone at G1 reduces ovulatory response by blunting GnRH-stimulated LH secretion [2].
Despite increased ovulation to G1, the proportion of cows with functional CL at PG1 was already high across all groups at ~96%, and no significant difference in serum progesterone concentrations at PG1 was found between the two GnRH treatments. Multiparous cows continued to show lower progesterone at PG1, reflecting higher milk production and associated hepatic metabolism. Furthermore, cows that ovulated to G1 had significantly greater increases in progesterone from G1 to PG1, confirming the formation of an accessory CL [2].
Ovulatory response to G2 was also improved in cows previously treated with 200 μg GnRH at G1 measuring at 93.8% vs. 89.8% with 100 μg, again with no effect of parity. This indicates better control of follicular dynamics across the protocol. Double ovulations at G2 remained more common in multiparous cows, likely due to their lower circulating P4 and greater LH pulsatility.
Pregnancy outcomes were favorably impacted by the higher GnRH dose. Cows receiving 200 μg had significantly higher pregnancy per AI at day 32 post-TAI measuring at 54.6%, than those receiving 100 μg that measured at 48.2%, with trends favoring the high dose at days 46, 88, and 200. However, cows treated with 200 μg had a tendency for slightly greater early pregnancy loss between days 32 and 46. No significant treatment and parity interaction was detected for pregnancies per AI [2].
Parity did affect pregnancy success: primiparous cows had higher pregnancies per AI at every time point compared to multiparous cows, consistent with previous research. Pregnancy loss was numerically greater in multiparous cows, potentially due to their higher rate of double ovulation, which is linked to twinning and early pregnancy failure.
Analysis also revealed that ovulation to G1 was a critical determinant of success across the protocol. Cows that ovulated to G1 had higher ovulatory response to G2, higher circulating progesterone at PG1, and a greater increase in progesterone from G1 to PG1. These cows also had significantly higher pregnancies per AI at all post-TAI time points, especially among primiparous animals. However, some cows that did not ovulate to G1 still achieved acceptable pregnancies per AI at a rate of >40%, indicating that ovulation to G1, while beneficial, is not a strict requirement for conception [2].
Additionally, it was noted that cows that ovulated to G1 had slightly lower GDPR than those that did not ovulate, contradicting expectations based on prior studies suggesting positive associations between genomic fertility scores and reproductive performance. This may reflect the known negative correlation between milk production and GDPR, as higher milk yield reduces progesterone, which in turn influences LH surge magnitude and ovulatory response. Thus, the interplay between milk yield, progesterone metabolism, and reproductive physiology may obscure direct associations between genomic fertility indices and outcomes in high-producing cows [2].
In conclusion, this study demonstrates that increasing the GnRH dose at G1 improves ovulatory responses, elevates progesterone concentrations, and enhances pregnancy outcomes without negatively affecting ovarian physiology. The data support using a higher GnRH dose to overcome high circulating progesterone and improve synchronization efficiency [2].

Disclaimer
**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Martínez M, Mapletoft RJ, Kastelic JP, Carruthers T. The effects of 3 gonadorelin products on luteinizing hormone release, ovulation, and follicular wave emergence in cattle. Can Vet J. 2003;44(2):125-131.

[2] Valdés-Arciniega TJ, Leão IMR, Anta-Galván E, et al. Effect of using 200 μg of gonadorelin at the first gonadotropin-releasing hormone of the breeding-Ovsynch on ovulatory response and pregnancies per artificial insemination in first-service lactating Holstein cows. J Dairy Sci. 2023;106(12):9718-9732. doi:10.3168/jds.2023-23416

Gonadorelin is a peptide that is similar to gonadotropin-releasing hormone (GnRH). GnRH is naturally made and released from the hypothalamus while gonadorelin is an injection that has been tested in cattle and rats. Gonadorelin has been shown to play a large role in the regulation of the reproductive system through the hypothalamus-pituitary gonadal (HPG) axis. When gonadorelin is administered it leads to the release of luteinizing hormone (LH) and follicle-stimulating hormone, which in turn, regulate the reproductive system in both males and females.

Effects of Gonadorelin on the Release of Luteinizing Hormone

In a study conducted by Martinez et. Al, three different variations of gonadorelin were given to cows in order to observe how the peptide would affect various follicular dynamics as well as the release of luteinizing hormone. Gonadorelin was formulated into three different variations for the sake of this study. In the preliminary experiment, nonlactating Holstein cows were randomly chosen to be given an intramuscular injection of gonadorelin diacetate tetrahydrate, the two forms being, Cytorelin (C) and Fertagyl (Fe). The cows then got their blood drawn for an LH analysis at 0, 1, 2, and 4 hours after injection in order to prepare for the second experiment.

In the second experiment, nonlactating Holstein cows were randomly chosen to be given injections of either Cytorelin, Fertagyl, or Factrel (FA, gonadorelin hydrochloride). The injection was given to the cows 6 to 7 days after their ovulation period and from there, an LH analysis was done 0,1, 2, 4, and 6 hours after treatment, and sonograms were performed twice a day to check for ovulation.

Following the second experiment, a third experiment was conducted with young, female beef cows where they were randomly selected to receive 1 of 3 of the gonadorelin formulation. From there the researchers waited 6 to 7 days after ovulation and took blood samples for LH analysis at hours 0, 0.5, 1, 1.5, 2, and 4 as well as sonograms of the ovaries.

Results of this study showed that in experiments 2 and 3, the Holstein cows treated with Cytorelin had higher mean and mean peak plasma LH concentrations. Furthermore, the Holstein cows treated with Cytorelin had a higher number of dominant follicles in an ovulation period than those treated with Fertagyl or Factrel. However, treatment with C versus FE or FA had an unremarkable effect on the beef heifers. Therefore, the study concluded that Cytorelin had the greatest effect on LH release (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC340045/).

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