A comparison of gonadotropin-releasing hormone and human chorionic gonadotropin in dairy cows with ovarian follicular cysts

a Knowledge Summary by

Katy Kesler DVM MS ^1*

Grace Longcore DVM ¹

Alex Russell DVM ¹

¹Michigan State University College of Veterinary Medicine, 736 Wilson Rd., East Lansing, MI 48824, USA
^*Corresponding Author (keslerka@msu.edu)

Clinical scenario

Cystic ovarian disease (COD) has been reported to occur at an incidence of between 6% and 30% of dairy cattle and is generally accepted as a common cause of reproductive failure in the dairy cow (Silvia et al., 2002; Ono et al., 2018; De Rensis et al., 2008; and Kesler & Garverick, 1982). Cystic ovarian disease is the presence of an anovulatory follicle ≥25 mm in diameter that persists for a minimum of 10 days in the absence of a functional corpus luteum (CL) (Ono et al., 2018; De Rensis et al., 2008; Garverick, 1997; and Kesler & Garverick, 1982). There are predominantly two classifications of ovarian cysts, follicular and luteal, distinguishable by the presence or absence of luteal tissue and circulating progesterone concentrations (Jeengar et al., 2014).  Cystic ovarian follicles have been reported to extend the calving interval by 20–64 days and, therefore, are of great economic importance to dairy producers (Silvia et al., 2002; and De Rensis et al., 2008). Moreover, the economic impact that results from the labour required for treatment and exogenous hormonal therapy for return to cyclicity cannot be overlooked. Many reports utilise exogenous hormones such as gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) for cyst luteinisation (Gundling et al., 2015 Rizzo et al., 2011; Kawate et al., 2011; and Nakao et al., 1980). This Knowledge Summary will address the question about which luteinising hormone results in a faster return to cyclicity.

You are a veterinarian performing routine herd health utilising ultrasound on a commercial dairy in the USA. The producer mentions that they are experiencing a high number of cystic cows on herd check. This producer asks you for recommendations regarding treating these cows; specifically, they want to get these cows cycling as quickly as possible.

The evidence

The literature search elicited six publications that met our criteria for inclusion. The six publications consisted of five randomised non-blinded comparative or controlled trials of level 3 (lower quality controlled trials [Howick et al., 2011]), (Taktaz et al., 2015; Mollo et al., 2012; Garverick et al., 1976; Elmore et al., 1975; and Nakao et al., 1992) and one randomised controlled trial of level 2 (well-designed controlled trial [Howick et al., 2011]), (Verma & Dabas, 1994). Additionally, many articles did not compare GnRH and hCG treatments directly or did not utilise time as a metric for evaluation and thus were excluded from this Knowledge Summary.

Dairy cattle were used in all of the evaluated articles, with 1/6 studies involving Holsteins, 2/6 studies involving crossbred cattle, 1/6 studies involving Friesians, and 2/6 studies involving Guernsey and Holstein cattle. All articles involved classification of ovarian follicular cyst size and persistence of at least one week; three articles evaluated cattle for the presence of luteal tissue either utilising ultrasound (Taktaz et al., 2015) or progesterone assays (Garverick et al., 1976; and Nakao et al., 1992). Cystic ovarian follicles were treated in all of the articles with GnRH and hCG therapy. The number of cows in each study ranged from relatively small studies with 20 cows to moderately sized comparisons, with 150 cows. The number of cows allocated for each treatment was highly variable per study ranging from 3–70 animals. Three studies had large treatment groups (30–70 animals per treatment) (Mollo et al., 2012; Taktaz et al., 2015; and Elmore et al., 1975), 1/6 studies had moderately sized treatment groups (17–18 animals per treatment) (Nakao et al., 1992), and 2/6 had very small treatment groups (10 animals per treatment) (Verma & Dabas,1994; and Garverick et al., 1976). Clinical cure (return to oestrus or cyst luteinisation), first oestrus conception, and interval to conception were consistently evaluated in the vast majority of studies. Additionally, many studies evaluated parameters such as recovery time, overall conception risk, pregnancy risk, breedings per conception, and interval to insemination.

In studies utilising a negative control group with no treatment, there was no significant difference in clinical response or subsequent fertility between GnRH and hCG, while both therapies outperformed the control group (Verma & Dabas, 1994). A higher percentage of animals treated with GnRH responded after one treatment than hCG; however, there were no significant differences in the number of animals that responded to either treatment or how quickly they responded to each treatment (Verma & Dabas, 1994). In studies that evaluated clinical cure as a return to oestrus, there were mixed results, with two studies showing hCG was more effective (Mollo et al., 2012; and Garverick et al., 1976) and two studies showing GnRH had more favourable results (Verma & Dabas, 1994; and Elmore et al., 1975). One study evaluated clinical cure as luteinisation of the cystic ovarian follicle (COF), and they found that hCG luteinised (14/17, 82.3%) 21.2% more cows than GnRH (11/18, 61.1%) (Nakao et al., 1992). Articles that studied interval to conception resulted in contradictory evidence, with two studies finding hCG treatment protocols required fewer days to conception (Taktaz et al., 2015; and Nakao et al., 1992) and two studies finding that GnRH had a shorter interval to conception (Garverick et al., 1976; and Elmore et al., 1975). Five out of six studies evaluated recovery time as days to first oestrus, and while there were no biologically or statistically significant differences, the majority of articles found that hCG required fewer days to first oestrus than GnRH treatment (Taktaz et al., 2015; Mollo et al., 2012; and Elmore et al., 1975), although one article had conflicting results (Garverick et al., 1976). Verma & Dabas (1994) listed their responses as a range of days to first oestrus for each treatment, and there was significant overlap between the GnRH treatment (15–30 days) and hCG treatment (17–31 days). Henceforth, there is still much research that needs to be conducted as to which treatment induces a more rapid return to cyclicity in dairy cattle with COD.

Many studies evaluated additional fertility parameters such as conception after first treatment or artificial insemination (AI), overall pregnancy or conception risks, and breedings per conception. Studies that evaluated conception after the first treatment or first AI had mixed results, with two articles indicating that hCG resulted in higher conception (Taktaz et al., 2015; and Mollo et al., 2012) and three articles suggesting GnRH had higher conception risks (Verma & Dabas, 1994; Elmore et al., 1975; and Nakao et al., 1992). When overall pregnancy risk was evaluated, Taktaz et al. (2015) at 70 days post treatment and Mollo et al. (2012) at 7 weeks post AI, found that hCG resulted in higher pregnancy risks; however, at 100 days, the Taktaz et al. (2015) group found that GnRH had a higher pregnancy risk. Moreover, when overall conception risk was evaluated, two groups found hCG to be more favourable (Elmore et al., 1975; and Nakao et al., 1992), while one group found GnRH to result in a higher conception risk (Garverik et al., 1976). Lastly, two studies evaluated the number of breedings per conception, while insignificant, both found that hCG treatment required more breedings per conception than GnRH treatments (Garverick et al., 1976; and Elmore et al., 1975).

Summary of the evidence

Appraisal, application and reflection

Ovarian follicular cysts are a common reproductive problem in the dairy industry, affecting approximately 6–30% of dairy cows (Silvia et al., 2002; Ono et al., 2018; De Rensis et al., 2008). However, the incidence of follicular cysts is likely underestimated, as 60% of the cysts that form before the first ovulation after freshening resolve without intervention (Ijaz et al., 1987; Jeengar et al., 2014; Kesler & Garverick, 1982). An ovarian follicular cyst has been described as a follicle that is 25 mm or larger in diameter and is persistent for at least 10 days in the absence of a CL (Garverick, 1997; Ono et al., 2018; De Rensis et al., 2008; Kesler and Garverick, 1982). Follicular cysts have been shown to add approximately 20 to 64 days to a dairy cow's open period (Silvia et al., 2002; De Rensis et al., 2008). Resulting in a significant negative economic impact for dairy producers, as a delay in conception leads to decreased milk yield, and resolution of the cyst often requires the expense of veterinary treatment.

The cause of ovarian follicular cysts is currently unknown (Vanholder et al., 2006, Amweg et al., 2013). There are several different treatment options for ovarian cysts, including manual rupture, aspiration, and hormonal injection.  Exogenous hormones are the most commonly used and successful form of treatment (Gundling et al., 2015 Rizzo et al., 2011, Kawate et al., 2011). Recently, manual rupture has become controversial due to results indicating that it may cause ovarian trauma that leads to the development of harmful haemorrhage and adhesions that can reduce fertility (Jeengar et al., 2014). Many reports argue the beneficial effects of gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) for treating ovarian luteal and follicular cysts (Taktaz et al., 2015). In addition, prostaglandin-F_2α (PGF_2α) has been shown to be effective against luteal cysts but not for follicular cysts. It is difficult to accurately determine whether a cyst is follicular or luteal by rectal palpation, and while accuracy is improved via ultrasound, progesterone assays remain the most definitive way of differentiating luteal and follicular cysts (Taktaz et al., 2015; Farin et al., 1992; Jeengar et al., 2014). Given the difficulty of distinguishing luteal and follicular cysts, Taktaz et al. (2015) suggested utilising a combination of PGF_2α and either GnRH or hCG to treat COD. However, Taktaz et al. (2015) did not find this combination therapy beneficial in shortening recovery time, interval to conception, conception risk, or pregnancy risk. Determining the most effective treatment method for ovarian follicular cysts is of utmost importance to dairy producers and veterinarians. The purpose of this Knowledge Summary is to evaluate the evidence comparing the length of time to return to ovarian cyclicity with the treatment of either GnRH or hCG.

The literature review resulted in six studies that met the inclusion criteria. Cystic ovarian follicles were identified utilising transrectal ultrasound or transrectal palpation. Mollo et al. (2012) evaluated recovery time, defined as the number of days between treatment and standing oestrus. Cattle were treated with GnRH (20 µg), hCG (3000 IU), or progesterone (1.55 g progesterone releasing intravaginal device for 10 days). However, the hCG group outperformed alternative treatments in every category (cure risk, pregnancy risk, and recovery time); no statistical or biologically significant differences were detected (Mollo et al., 2012). These results are further supported by Taktaz et al. (2015), who reported an insignificant 1.6 day reduction in recovery time with hCG (1500 IU) compared to conventional treatment (GnRH analogue) in cows with follicular cysts. However, previous literature has suggested conflicting results, with Garverick et al. (1976) indicating cattle treated with GnRH had a substantially shorter interval to conception (59.7 ± 9 vs 91 ± 23 days) and required 2.7 fewer days from treatment to first oestrus or ovulation when compared to hCG treatment. Additionally, a recent study has suggested that cattle treated with hCG ovulate significantly later than cattle treated with GnRH, 4.9 hours on average regardless of dioestrus periods (Liu et al., 2019). Moreover, Elmore et al. (1975) found that cattle treated with hCG required 1.9 fewer days from treatment to first oestrus; however, the interval from treatment to conception was 10.4 days longer than their GnRH-treated counterparts. This may be partially due to the fact that hCG-treated cattle, on average, required more breedings per conception than the GnRH-treated group. Henceforth, there is still much debate amongst the scientific community on which treatment induces a more rapid return to cyclicity in dairy cattle afflicted with COD.

It has been suggested that low-circulating progesterone concentrations may have a role in the pathogenesis of COFs due to the role of progesterone in regulating the effects of oestradiol (Wiltbank et al., 2002). Understandably, many publications have investigated luteal profiles (progesterone concentrations and CL diameter), the number of CL present on the ovary, circulating progesterone concentrations, as well as the effects of hCG and GnRH on follicular luteinisation. There was no clear consensus on the effects of hCG and GnRH on the aforementioned parameters. While two articles suggested that there was no significant difference in circulating progesterone concentrations (Yotov et al., 2014 Nakao et al., 1980), another article indicated that cattle treated with hCG (3000 IU) had significantly higher plasma progesterone levels suggesting that hCG may be more effective at luteal phase induction (Singh et al., 2012). Moreover, Singh et al. (2012) found that post-AI luteal profiles were significantly more favourable in hCG treated cattle; it was proposed that this could be the result of elevated pre-conception plasma progesterone concentrations.

The efficacy of GnRH and hCG in treating bovine ovarian follicular cysts should be further evaluated with more controlled studies. Most of the articles found during the literature review for this Knowledge Summary were small studies that did not include a control group receiving no therapeutics in the study design. Further analysis to ensure that spontaneous resolution of ovarian cysts that occur naturally are accounted for in the study design or with statistical modeling would be beneficial; since the rate of spontaneous cure has been reported to be as high as 60% in fresh cows that have not yet ovulated (Ijaz et al., 1987; Jeengar et al., 2014; Kesler and Garverick, 1982). Some articles achieved this by comparing the proportion of animals in each treatment group relative to their days in milk (DIM), suggesting that if proportions of cows less than 40 DIM were equivocal, then the spontaneous cure rates should not be impacting results (Taktaz et al., 2015). Moreover, a network analysis comparing the efficacy of treatment methods may be beneficial. However, due to the heterogeneity of the studies identified, at this time, the treatments are unequivocal, and network analysis cannot be performed (Lean et al., 2009). For example, many studies were excluded as they did not directly compare GnRH and hCG treatments; Nakao et al. (1979) performed a small crossover trial where animals were enrolled in subsequent treatment groups when they failed to respond to previous therapy, making it challenging to differentiate subsequent treatment failures from individual differences in fertility.

To provide more guidance for the clinical recommendation and treatment of ovarian follicular cysts, the route of administration of the chosen therapeutics should also be further investigated. Rizzo et al. (2011) compared the intramuscular and epidural use of lecirelin, a GnRH analogue, to treat bovine ovarian follicular cysts. They found that epidural administration of lecirelin in cows with follicular cysts resulted in a higher percentage of cows returning to oestrus than those cows which received an intramuscular injection of lecirelin (Rizzo et al., 2011). This effect was attributed to the action of lecirelin on GnRH receptors in the spinal cord and ovary, and possibly on the pituitary gland directly through the epidural canal (Rizzo et al., 2011). These findings support the possibility of further improvements in the treatment of bovine ovarian follicular cysts by altering existing treatment methods.

Moreover, studies to determine the safest and most efficacious dose of hCG may also be beneficial. Among the studies in this Knowledge Summary, the dose of hCG administered to cows with ovarian follicular cysts ranged from 1,500 IU (Taktaz et al., 2015) to 3,000 IU (Mollo et al., 2012; Verma & Dabas, 1994; Garverick et al., 1976; Nakao et al., 1992) to as high as 10,000 IU hCG (Elmore et al., 1975). Other reports have published dosages ranging from 500 IU (Yotov et al., 2014 to 20,000 IU (Nakao et al., 1979). Human chorionic gonadotropin has a high molecular weight and carbohydrate content and thus can lead to the production of hCG antibodies and anaphylaxis in cattle (De Rensis et al., 2010; Giordano et al., 2012). To decrease the likelihood of administering an excessive amount of hCG and causing an immune response, the minimum effective dose should be established to guide future treatment plans. Alternatively, treatment with hCG may be divided into multiple smaller doses of hCG, as this has been shown to be equally effective as a single large dose in modulating ovarian follicular development in dairy cows with cystic ovarian follicles (Ono et al., 2018).

As a final note, it was indicated in the literature that hCG, which historically was the treatment of choice for COD and for unknown reasons, became challenging to acquire and cost-prohibitive for producers (Seguin et al., 1976). Seguin et al. (1976) suggested cost increases were due to challenges in obtaining hCG as it is isolated from pregnant women's urine and speculated that the synthetic molecule GnRH would provide a more cost-effective and stable supply chain (Seguin, 1976). Moreover, Ngategize et al. (1987) found that it was more economical to treat cattle with GnRH than with hCG; however, both treatment options were found to be more economically advantageous than no treatment or spontaneous resolution (Ngategize et al., 1987). This conclusion was based on assumptions that GnRH resulted in the more successful resolution of COF, cost less than hCG, but required a longer recovery (Ngategize et al., 1987). Regardless, Ngategize et al. (1987) suggest that days to first oestrus (recovery time) would need to decrease by 50% for hCG to compete out GnRH therapy (Ngategize et al., 1987).

In conclusion, at this time, there is insufficient evidence to suggest whether GnRH or hCG is a more efficacious treatment for ovarian follicular cysts, and further research is required to elucidate which treatment results in a more rapid return to cyclicity for cattle afflicted with cystic ovarian follicles. Without additional evidence when considering practical implementation in field conditions, GnRH is a more appropriate first-line therapeutic for cystic ovarian disease due to challenges surrounding the immunogenicity, availability, and cost of hCG therapy.

Methodology Section

Search Strategy
Databases searched and dates covered:	CAB Abstracts database accessed on WOS platform (1973–2021) PubMed Central accessed on NCBI website (1910–2021)
Search strategy:	CAB Abstracts: (dairy OR lactating OR cow OR cattle OR heifer OR bovine) AND ((cystic OR cysts) AND ovarian AND (disease OR follicle OR follicular)) (Reproductive performance OR ovulation OR estrus OR conception OR pregnancy OR preg rate OR oestrus) (human chorionic gonadotropin OR hCG) AND (gonadotropin releasing hormone OR GnRH) 1 AND 2 AND 3   PubMed: (dairy OR lactating OR cow OR cattle OR heifer OR bovine) AND ((cystic OR cysts) AND ovarian AND (disease OR follicle OR follicular)) AND (Reproductive performance OR ovulation OR estrus OR conception OR pregnancy OR preg rate OR oestrus) AND (human chorionic gonadotropin OR hCG) AND (gonadotropin releasing hormone OR GnRH)
Dates searches performed:	20 Dec 2021

Exclusion / Inclusion Criteria
Exclusion:	Non-English language, non-primary literature, non-bovine, studies not relevant to the PICO question.
Inclusion:	Use of GnRH and hCG for the treatment of ovarian follicular cysts, time as a metric for evaluation.

Search Outcome
Database	Number of results	Excluded – Non-English language publication	Excluded – Non-primary literature	Excluded – Non-bovine	Excluded – Not relevant to PICO question	Total relevant papers
CAB Abstracts	67	21	17	5	19	5
PubMed	20	4	7	3	5	1
Total relevant papers when duplicates removed						6

The authors declare no conflicts of interest.

The authors are veterinary students at Michigan State University (East Lansing, MI, USA) that completed this Knowledge Summary as part of the LCS 679 Food Animal Production Medicine 1 course.

1. Amweg, A. N., Salvetti, N. R., Stangaferro, M. L., Paredes, A. H., Lara, H. H., Rodríguez, F. M. & Ortega, H. H. (2013). Ovarian localization of 11β-hydroxysteroid dehydrogenase (11βHSD): effects of ACTH stimulation and its relationship with bovine cystic ovarian disease. Domestic Animal Endocrinology. 45(3), 126–140. DOI: https://doi.org/10.1016/j.domaniend.2013.07.001
2. De Rensis, F., Bottarelli, E., Battioni, F., Capelli, T., Techakumphu, M., García-Ispierto, I. & López-Gatius, F. (2008). Reproductive performance of dairy cows with ovarian cysts after synchronizing ovulation using GnRH or hCG during the warm or cool period of the year. Theriogenology. 69(4), 481–484. DOI: https://doi.org/10.1016/j.theriogenology.2007.10.018
3. De Rensis, F., López-Gatius, F., Garcia-Ispierto, I. & Techakumpu M. (2010). Clinical use of human chorionic gonadotropin in dairy cows: an update. Theriogenology. 73(8), 1001–1008. DOI: https://doi.org/10.1016/j.theriogenology.2009.11.027
4.  Elmore, R. G., Bierschwal, C. J., Youngquist, R. S., Cantley, T. C., Kesler, D. J. & Garverick, H. A. (1975). Clinical responses of dairy cows with ovarian cysts after treatment with 10,000 IU HCG or 100 mcg GnRH. Veterinary Medicine, Small Animal Clinician. 70(11), 1346–1349.
5. Farin, P. W., Youngquist, R. S., Parfet, J. R. & Garverick, H. A. (1992). Diagnosis of luteal and follicular ovarian cysts by palpation per rectum and linear-array ultrasonography in dairy cows. Journal of the American Veterinary Medical Association. 200(8), 1085–1089.
6. Garverick, H. A., Kesler, D. J., Cantley, T. C., Elmore, R. G., Youngquist, R. S. & Bierschwal, C. J. (1976). Hormone response of dairy cows with ovarian cysts after treatment with HCG or GnRH. Theriogenology. 6(4), 413–425. DOI: https://doi.org/10.1016/0093-691X(76)90108-4
7. Garverick, H. A. (1997). Ovarian follicular cysts in dairy cows. Journal of Dairy Science. 80(5), 995–1004. DOI: https://doi.org/10.3168/jds.S0022-0302(97)76025-9
8. Giordano, J. O., Wiltbank, M. C. & Fricke, P. M. (2012). Humoral immune response in lactating dairy cows after repeated exposure to human chorionic gonadotropin. Theriogenology. 78(1), 218–224. DOI: https://doi.org/10.1016/j.theriogenology.2012.02.003
9. Gundling, N., Drews, S. & Hoedemaker, M. (2015). Comparison of two different programmes of ovulation synchronization in the treatment of ovarian cysts in dairy cows. Reproduction in Domestic Animals. 50(6), 893–900. DOI: https://doi.org/10.1111/j.1439-0531.2009.01342.x
10. Howick, J., Chalmers, I., Glasziou, P., Greenhalgh, T., Heneghan, C., Liberati, A., Moschetti, I., Phillips, B. & Thornton, H. (2011). The 2011 Oxford CEBM evidence levels of evidence (introductory document). Oxford Center for Evidence Based Medicine. [online] Available from: https://www.cebm.ox.ac.uk/resources/levels-of-evidence/ocebm-levels-of-evidence [Accessed 05 Aug 2021].
11. Ijaz, A., Fahning, M. L. & Zemjanis, R. (1987). Treatment and control of cystic ovarian disease in dairy cattle: a review. British Veterinary Journal. 143(3), 226– DOI: https://doi.org/10.1016/0007-1935(87)90085-6
12. Jeengar, K., Chaudhary, V., Kumar, A., Raiya, S., Gaur, M. & Purohit, G. N. (2014). Ovarian cysts in dairy cows: old and new concepts for definition, diagnosis and therapy. Animal Reproduction. 11(2), 63–73.
13. Kawate, N., Watanabe, K., Uenaka, K., Takahashi, M., Inaba, T. & Tamada, H. (2011). Comparison of plasma concentrations of estradiol-17β and progesterone, and conception in dairy cows with cystic ovarian diseases between Ovsynch and Ovsynch plus CIDR timed AI protocols. Journal of Reproduction and Development. 57(2), 267–272. DOI: https://doi.org/10.1262/jrd.10-066t
14. Kesler, D. J. & Garverick, H. A. (1982). Ovarian cysts in dairy cattle: a review. Journal of Animal Science. 55(5), 1147–1159. DOI: https://doi.org/10.2527/jas1982.5551147x
15. Lean, I. J., Rabiee, A. R., Duffield, T. F. & Dohoo, I. R. (2009). Invited review: Use of meta-analysis in animal health and reproduction: Methods and applications. Journal of Dairy Science. 92(8), 3545–3565. DOI: https://doi.org/10.3168/jds.2009-2140
16. Liu, T. C., Ho, C. T., Li, K. P., Chang, C. C. & Chan, J. P. W. (2019). Human chorionic gonadotropin (hCG)-induced ovulation occurs later but with equal occurrence in lactating dairy cows: comparing hCG and gonadotropin-releasing hormone protocols. Journal of Reproductive Development. 65(6): 507–514. DOI: https://doi.org/10.1262/jrd.2019-037
17. Mollo, A., Stradaioli, G., Gloria, A. & Cairoli, F. (2012). Efficacy of different ovarian cysts treatments (GnRH, hCG and PRID) in dairy cows. Journal of Animal and Veterinary Advances. 11(21), 4058–4063. DOI: https://doi.org/10.3923/javaa.2012.4058.4063
18. Nakao, T., Tsurubayashi, M., Horiuchi, S., Nomura, T., Ishibashi, Y., Kubo, M. & Kawata, K. (1979). Effects of a systematic application of human chorionic gonadotropin, gonadotropin-releasing hormone analog and bovine anterior pituitary gonadotropin in cows with cystic ovarian disease. Theriogenology. 11(5), 385–397. DOI: https://doi.org/10.1016/0093-691X(79)90062-1
19. Nakao, T., Kawata, K. & Numata Y. (1980). Therapeutic effects of an analog of luteinizing hormone-releasing hormone (Des-Gly¹⁰-LH-RH-Ethylamide) on cows with cystic ovary. The Japanese Journal of Veterinary Science. 42(4), 459–462. DOI: https://doi.org/10.1292/jvms1939.42.459
20. Nakao, T., Tomita, M., Kanbayashi, H., Takagi, H., Abe, T., Takeuchi, Y., Ociai, H., Moriyoshi, M. & Kawata, K. (1992). Comparisons of several dosages of a GnRH analog with the standard dose of hCG in the treatment of follicular cysts in dairy cows. Theriogenology. 38(1), 137–145. DOI: https://doi.org/10.1016/0093-691X(92)90225-G
21. Ngategize, P. K., Kaneene, J. B., Harsh, S. B., Bartlett, P. C. & Mather, E. L. (1987). A financial analysis of alternative management strategies of cystic follicles. Preventive Veterinary Medicine. 4(5–6), 463–470. DOI: https://doi.org/10.1016/0167-5877(87)90031-6
22. Ono, T., Takagi, M., Kawashima, C., Wijayagunawardane, M. P. B., Vos, P. L. A. M., Taniguchi, M. & Otoi, T. (2018). Comparative effects of different dosages of hCG on follicular development in postpartum dairy cows with cystic ovarian follicles. Frontiers in Veterinary Science. 5, 130. DOI: https://doi.org/10.3389/fvets.2018.00130
23. Rizzo, A., Campanile, D., Mutinati, M., Minoia, G., Spedicato, M. & Sciorsci, R. L. (2011). Epidural vs intramuscular administration of lecirelin, a GnRH analogue, for the resolution of follicular cysts in dairy cows. Animal Reproduction Science. 126(1–2), 19–22. DOI: https://doi.org/10.1016/j.anireprosci.2011.04.013
24. Seguin, B. E., Convey, E. M. & Oxender, W. D. (1976). Effect of gonadotropin-releasing hormone and human chorionic gonadotropin on cows with ovarian follicular cysts. American Journal of Veterinary Research. 37(2), 153–157.
25. Silvia, W. J., Hatler, T. B., Nugent, A. M. & Da Fonseca, L. L. (2002). Ovarian follicular cysts in dairy cows: an abnormality in folliculogenesis.Domestic Animal Endocrinology. 23(1–2), 167–177. DOI: https://doi.org/10.1016/S0739-7240(02)00154-6
26. Singh, J., Ghuman, S. P. S. & Honparkhe, M. (2012). Reproductive performance following GNRH or HCG and PGF2α administration in crossbred cattle with ovarian cysts. Veterinary Practitioner. (1), 57–59.
27. Taktaz, T., Kafi, M., Mokhtari, A. & Heidari, M. (2015). Reproductive responses of dairy cows with ovarian cysts to simultaneous human chorionic gonadotropin or gonadotropin-releasing hormone and cloprostenol compared to gonadotropin-releasing hormone alone treatment. Veterinary World. 8(5), 640–644. DOI: https://doi.org/10.14202/vetworld.2015.640-644
28. Vanholder, T., Opsomer, G. & De Kruif, A. (2006). Aetiology and pathogenesis of cystic ovarian follicles in dairy cattle: a review. Reproduction Nutrition Development. 46(2), 105–119. DOI: https://doi.org/10.1051/rnd:2006003
29. Verma, M.C. & Dabas, Y.P.S. (1994). Treatment of ovarian cysts in cows. Indian Journal of Animal Reproduction: Journal of the Indian Society for the Study of Animal Reproduction. 15(2), 125–126.
30. Wiltbank, M. C., Gümen, A. & Sartori, R. (2002). Physiological classification of anovulatory conditions in cattle. Theriogenology. 57(1), 21–52. DOI: https://doi.org/10.1016/s0093-691x(01)00656-2
31. Yotov, S. A., Atanasov, A. S., Georgiev, G. B., Dineva, J. D. & Palova, N. A. (2014). Investigation on some biochemical parameters and effect of hormonal treatment in anoestrous dairy cows with cystic ovarian follicle. Asian Pacific Journal of Reproduction. 3(1), 41–45. DOI: https://doi.org/10.1016/S2305-0500(13)60183-9