- Email: [email protected]
Using Estrous Behavior to Time Initiation of Oxytocin Administration to Prolong Luteal Function in Mares
Journal of Equine Veterinary Science 75 (2019) 78e81
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Using Estrous Behavior to Time Initiation of Oxytocin Administration to Prolong Luteal Function in Mares Hannah S. Manning, Erin E. Runcan*, Christa R. Dias de Moraes, Marco A. Coutinho da Silva Department of Veterinary Clinical Sciences, The Ohio State University College of Veterinary Medicine, Columbus, OH
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2018 Received in revised form 29 January 2019 Accepted 30 January 2019 Available online 6 February 2019
The objective of this study was to use estrous behavior alone to determine the appropriate time for beginning an oxytocin treatment protocol for estrus suppression. We hypothesized that administration of oxytocin beginning 8 days after the onset of estrus will prolong the luteal phase in mares. Twentythree light breed mares (aged 4e20 years) were exposed to a stallion and observed for signs of sexual receptivity. Mares not displaying signs received 250 mg of cloprostenol intramuscularly (IM) and were teased again 3e4 days later. On the day that estrous behavior was observed (Day 0), mares were randomly divided into two groups: oxytocin (n ¼ 11): oxytocin (60 IU, IM) was administered once daily from Day 8e17; control (n ¼ 12): did not receive treatment. Blood was collected from all mares every 4 days throughout Day 17, and every 7 days thereafter until Day 45. Serum progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. Interestrus interval was defined as the period between Day 0 and the day when progesterone next reached <1.0 ng/mL. The average interestrus interval was higher for treated mares compared with control mares (32.4 ± 4.2 vs. 21.8 ± 1.5 days, respectively, P ¼ .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 (54.5%) mares and up to 45 days in 5 of 11 mares (45.5%). We conclude that luteal maintenance beyond 30 days was attained by once-daily oxytocin administration beginning 8 days following behavioral estrus in a majority of mares. © 2019 Elsevier Inc. All rights reserved.
Keywords: Horse Oxytocin Mare Estrus Behavior Corpus luteum
1. Introduction Poor performance due to undesirable estrous behavior is a common complaint made by owners and trainers of performance mares. Typically, the mare is in a period of estrus or sexual receptivity toward the stallion from 5 to 7 days during the estrous cycle. Such estrous behaviors range from benign tail swishing, squealing, and frequent urination to more severe behavior such as poor attitude, aggressiveness, and difficulty handling and training [1]. Because these behaviors can be very problematic for performance horse owners and trainers, veterinarians are often consulted to determine ways to suppress estrus and eliminate the undesirable
Animal welfare/ethical statement: All animal use and procedures were approved by The Ohio State University, Institutional Animal Care and Use Committee, IACUC #2016A00000008. Conflict of interest statement: The authors declare no conflicts of interest. * Corresponding author at: Erin E. Runcan, Department of Veterinary Clinical Sciences, The Ohio State University College of Veterinary Medicine, 601 Vernon L. Tharp St., Columbus, OH 43210. E-mail address: [email protected] (E.E. Runcan). https://doi.org/10.1016/j.jevs.2019.01.012 0737-0806/© 2019 Elsevier Inc. All rights reserved.
behavior. Multiple methods may be used to suppress estrus including hormonal therapy with progesterone, intrauterine devices, and in extreme cases ovariectomy [1,2]. Hormonal suppression of estrus with progesterone has been a known, effective method of estrus suppression for many years, and there are many different formulations. Natural progesterone in an oil base can be administered intramuscularly once daily. However, this method of administration can result in local tissue inflammation and pain as a result of the repeated injections. It is therefore not commonly used for suppression of estrus in performance mares [3]. Long-acting progesterone preparations have also been reported to be efficacious for estrus suppression. This preparation is administered once via an injection intramuscularly. There are 12day and 30-day formulations available; however, these longacting preparations may not be as effective as once-daily treatment and also have the ability to cause muscle soreness on injection [1]. Synthetic progesterone products are another method of estrus suppression. Altrenogest is the only highly effective and safe progestin approved for use in mares. It is dosed orally and administered once daily. Although very effective, treatment must be administered 3e4 days before an event and continued daily
H.S. Manning et al. / Journal of Equine Veterinary Science 75 (2019) 78e81
throughout the showing period [1]. Owners and trainers administering altrenogest are also at an increased risk, as it can have adverse health effects with inappropriate human exposure [4]. Veterinarians have also suppressed estrus in mares with the use of an intrauterine glass marble. Studies have shown this method is inexpensive and easy to administer; however, the efficacy is limited (41.7%) [5]. There have also been many risks reported in association with the use of glass marbles. For example, the prolonged presence of a glass marble has been associated with pyometra, chronic intermittent colic, and even the establishment of pregnancy and subsequent abortion. There is also a potential for harm to the mare, should the glass marble be damaged during removal or while inside of the uterus [6]. Finally, for extreme cases, ovariectomy is another method used to suppress estrus. This method would ultimately remove the source of the hormones producing aberrant estrous behavior. However, this method is considered a last resort, as it is permanent and irreversible [1]. Surgical complications, although uncommon, are possible and include hemorrhage, infection, adhesions, herniation, and evisceration. The procedure is also not 100% effective, with a small percentage (20%) of mares continuing to exhibit estrus-like behaviors postovariectomy in one study [7]. Previous research has shown that administration of exogenous oxytocin from Days 7e14, during diestrus, is a very effective, lowcost, and practical method of suppressing estrus [8,9]. The protocol works by inhibiting luteolysis, extending function of the corpus luteum, and maintaining elevated levels of the steroid hormone progesterone in the bloodstream, which suppresses behavioral estrus. The mechanism of prolonged function of the corpus luteum via exogenous oxytocin administration before Day 10 has been shown to be a result of oxytocin preventing the upregulation of cyclooxygenase-2 expression in the endometrium causing subsequent impairment of PGF2a secretion and synthesis [10,11]. Vanderwall et al reported luteostasis in 100% of horses treated in this initial “proof of principle study” [8]. The researchers then went on to report 60%e70% efficacy in prolonging the corpus luteum when evaluating once-daily versus twice daily-oxytocin administration in a larger population of mares [12]. Although exogenous oxytocin administration to suppress estrus is successful, it currently requires serial costly veterinary visits to determine the exact date of ovulation. The use of transrectal ultrasonography and palpation to detect ovulation and estrus in the mare must occur before the protocol can begin. Thus, the present study aims to eliminate such veterinary visits, as it may not be feasible in all situations. The objective of this study was to determine whether oxytocin administration beginning at Day 8 after behavioral onset of estrus will be effective in prolonging the luteal phase in mares, without knowing the exact date of ovulation.
79
signs used to indicate sexual receptivity included posturing toward the stallion, squatting to urinate, winking, and so on. When mares were teased, they were designated positive, passive, or negative. If mares did not show any signs of sexual receptivity (squealing, kicking, pinning of the ears, tail clamping, or actively moving away from the stallion), they were considered negative. Luteolysis was induced (250 mg Cloprostenol, intramuscularly (IM); Merck Animal Health, Madison, NJ) for these mares, and teasing was resumed 3e4 days later. If mares teased passively (standing quietly, allowing the stallion to touch without kicking, ears neutral), they were continued to be teased once daily until they showed more positive indicators of estrus. On the first day mares teased positive or exhibited behavioral estrus (Day 0), mares were randomly assigned to an experimental group (n ¼ 11) or control group (n ¼ 12). The control group did not receive treatment and was used to confirm a normal estrous cycle and progesterone profile. In the experimental group, mares received 60 IU of Oxytocin IM (right or left neck; Bimeda e MTC Animal Health, Cambridge, Ontario, Canada) once a day starting on Day 8 and continuing until Day 17. Treatment was initiated on Day 8 to account for a normal estrus period of 5e7 days and allow sufficient time for ovulation to occur. Beginning on Day 0, jugular blood samples (10 mL) were drawn via a vacutainer from experimental and control groups every 4 days through Day 17 and every 7 days thereafter until Day 45. The experiment was conducted through Day 45 to allow time to demonstrate efficacy of the protocol beyond the duration of a normal physiological estrous cycle and still remain within the time constraints of the study. 2.3. Progesterone Assay Blood samples were allowed to coagulate and then centrifuged at 2000 g for 10 minutes. The serum was drawn off of the samples and frozen at 80 C until analysis. Samples were run in house at The Ohio State University Veterinary Hospital using a Siemens/DPC Immulite Analyzer (The Ohio State University, Columbus, OH) to determine progesterone concentration. Progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. 2.4. Statistical Analysis
2. Materials and Methods
The interestrus interval was defined as the period between Day 0 and the day when serum progesterone next reached <1.0 ng/mL. The average interestrus interval between groups was compared by independent samples t-test using Excel's statistical software package. Data are presented as mean ± standard error of the mean. Significance was set at P < .05. The proportion of extended interestrus intervals between groups was compared using Fisher's Exact Test.
2.1. Animals
3. Results
This study was conducted in the northern hemisphere between the months of May and October. Twenty-three light breed mares, 4e20 years of age and owned by The Ohio State University College of Veterinary Medicine for teaching and research, were used for this study. All animal procedures were approved and conducted according to the guidelines of The Ohio State University Institutional Animal Care and Use Committee.
The average interestrus interval was higher for oxytocin treated mares compared with the control group, 32.4 ± 4.2 versus 21.8 ± 1.5 days, respectively (P ¼ .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 mares (54.5%) and up to 45 days in 5 of 11 mares (45.45%; Figs. 1A and 1B). Progesterone levels oscillated normally in 11 of 12 control mares. Two control mares were enrolled in the study but were not truly in estrus, made evident by progesterone concentrations >1.0 ng/mL at Day 0. One control mare experienced a spontaneously prolonged luteal phase of her cycle, which is evident by persistently elevated progesterone beyond 25 days (Fig. 2). When comparing 1 of 12 control
2.2. Experiment Twenty-three light breed mares were teased to a stallion and observed for signs of sexual receptivity or behavioral estrus. The
H.S. Manning et al. / Journal of Equine Veterinary Science 75 (2019) 78e81
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Fig. 1. (A) Blood progesterone levels from mares treated with oxytocin and resultant prolongation of luteal function. The red line indicates the progesterone reference (1.0 ng/mL) used to determine whether mares had a functioning corpus luteum (>1.0 ng/mL indicated a functioning CL). Day 0 was the first day mares displayed behavioral signs of estrus. (B) Blood progesterone levels from mares treated with oxytocin that did not result in prolongation of luteal function. The red line indicates the progesterone reference (1.0 ng/mL) used to determine whether mares had a functioning corpus luteum (>1.0 ng/mL indicated a functioning CL). Day 0 was the first day mares displayed behavioral signs of estrus.
4. Discussion
mares with a naturally extended interestrus interval with 6 of 11 experimental mares with an extended interestrus interval, the onetailed P value equaled .02. The comparison between 1 of 12 control mares and 5 of 11 experimental mares that had an extended luteal phase beyond 45 days yielded a one-tailed P value of .06.
These results confirm that administration of exogenous oxytocin on Days 8e17 post behavioral onset of estrus was efficacious in 54.5% of mares. To ensure treatment was based on behavior alone,
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Fig. 2. Blood progesterone levels from mares that did not receive oxytocin treatment and served as controls. The gray line represents the mare with suspected retained corpus luteum. The red line indicates the progesterone reference (1.0 ng/mL) used to determine whether mares had a functioning corpus luteum (>1.0 ng/mL indicated a functioning CL). Day 0 was the first day mares displayed behavioral signs of estrus.
H.S. Manning et al. / Journal of Equine Veterinary Science 75 (2019) 78e81
transrectal ultrasonographic evaluation was not performed to document ovulation. As described in previous work by Vanderwall et al [12], efficacy of this treatment protocol would be improved by documenting that ovulation had indeed occurred rather than waiting a set time of 8 days from the appearance of behavioral estrus. When comparing the extended interestrus intervals to 30 days, there was a significant difference between the one control mare and six experimental mares that had a prolonged luteal phase. When evaluating the group that had an extended luteal phase beyond 45 days, 1 of 12 control mares compared with 5 of 11 experimental mares was not statistically significant (P ¼ .06); however, there was clearly a positive trend noted in this group of mares. This result would have likely been significant had a larger treatment group been evaluated. Because treatment enrollment was based on estrous behavior alone, variability exists as to the exact day in estrus that defines “Day 0.” Because of this, it is not surprising that some mares did not adequately respond. In previous work by Rebordao et al [11], it has been noted that oxytocin is antiluteolytic when treatment is initiated before Day 10 of diestrus and proluteolytic when administered after Day 14. In our subset of mares that did not respond to treatment, it is apparent that the initial injections of oxytocin began too early in diestrus to be effective, which is evident by the lack of elevated progesterone at the time of oxytocin administration. Our results are lower than reported success rates obtained in previous studies that initiated oxytocin treatments based on the day of ovulation (60%e70%, 100%) [8,12]. The same authors reported improved efficacy when oxytocin treatment duration was extended up to 29 days [12]. Keith et al also reported increased efficacy of oxytocin treatment with increasing number of treatment days (Days 8e10 vs. Days 8e12 vs. Days 8e14 postovulation) [10]. Potentially, the success rate of the present study would be improved if oxytocin treatment was initiated further into diestrus, and the duration of treatment was extended beyond 10 days. This would provide a longer duration of treatment to ensure continuation through the end of diestrus, especially because the date of ovulation is unknown. For the control group (Fig. 2), 11 of the 12 mares had a normal estrous cycle and progesterone profile. A subset of control mares depicted in Fig. 2 was only evaluated for 31 days because of time constraints where they were housed. We suspect the mare with elevated progesterone (>1.0 ng/mL) throughout her cycle had a prolonged luteal phase either from a retained corpus luteum or a diestrus ovulation. The phenomena of a persistent luteal phase are relatively common in the mare, occurring in up to 25% of documented equine estrous cycles [13]. The exact reason for the persistently elevated progesterone in this control mare is unknown, as ovarian structures were not monitored through the study. In our study, we used progesterone concentrations alone to determine the persistence of the luteal phase in experimental mares, indicated by serum progesterone above 1.0 ng/mL. Although estrous behavior was not assessed throughout the study, it is expected that mares with progesterone concentrations above 1.0 ng/ mL would not show estrous behavior. Based on serum progesterone concentrations alone, mares with progesterone concentrations above 1.0 ng/mL should have an active corpus luteum and should therefore not show estrous behavior [14]; however, it is important to note that not all mares show the same behavioral indicators of estrus. Some mares showed fairly subtle signs, making the use of
81
behavior alone for determining when to initiate oxytocin treatment challenging. We suspect this is why two of the control mares were enrolled in the protocol that were not truly in estrus as depicted by progesterone concentrations >1.0 ng/mL on Day 0. 5. Conclusion Luteal maintenance beyond 31 days was attained in 54.5% of mares by once-daily oxytocin administration beginning 8 days following behavioral signs of estrus. The outcomes of this study are relevant to cases in which multiple veterinary visits (including transrectal palpation and ultrasonography) may not be feasible in all situations. One major limitation of this protocol is the requirement that either a stallion be present on farm to evaluate behavior or a veterinary examination be performed to confirm the mare is in fact in estrus at the time the aberrant behavior is noted. Further research is needed to improve efficacy of this protocol. We believe that efficacy may be improved by beginning treatment later into diestrus (beyond Day 8) and increasing the treatment duration. With modifications and further research, we believe this protocol could be a practical and low-cost method of estrus suppression for performance horse owners and trainers. Financial Disclosure This work was supported by The Ohio State University Veterinary Scholars Summer Research Program. References [1] McCue PM. Estrus suppression in performance horses. J Equine Vet Sci 2003;23:342e4. [2] McCue PM. Hormone therapy in clinical equine practice. Vet Clin North Am Equine Pract 2016;32:425e34. [3] Loy RG, Swan SM. Effects of exogenous progestogens on reproductive phenomena in mares. J Anim Sci 1966;25:821e6. [4] Fact Sheet, Regu-Mate®. Intervet Inc., Merck Animal Health; 2012. https:// www.merck-animal-health-usa.com/pdfs/equine/Regu-Mate%20-Fact%20Sheet %20-%20Merck%20Animal%20Health.pdf. [Accessed 15 February 2019]. [5] Nie GJ, Johnson KE, Braden TD, Wenzel JGW. Use of an intra-uterine glass ball protocol to extend luteal function in mares. J Equine Vet Sci 2003;23:266e73. [6] Vanderwall DK. Is it time to retire the use of intrauterine glass balls for estrus suppression in mares? J Am Vet Med Assoc 2015;247:346e7. [7] Roessner HA, Kurtz KA, Caron JP. Laparoscopic ovariectomy diminishes estrusassociated behavioral problems in mares. J Equine Vet Sci 2015;35:250e3. [8] Vanderwall DK, Rasmussen DM, Woods GL. Effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares. J Am Vet Med Assoc 2007;231:1864e7. [9] Vanderwall DK, Rasmussen DM, Carnahan KG, Davis TL. Effect of administration of oxytocin during diestrus on corpus luteum function and endometrial oxytocin receptor concentration in cycling mares. J Equine Vet Sci 2012;32:536e41. [10] Keith L, Ball BA, Scoggin K, Esteller-Vico A, Woodward ET, Troedsson MHT, et al. Diestrus administration of oxytocin prolongs luteal maintenance and reduces plasma PGFM concentrations and endometrial COX-2 expression in mares. Theriogenology 2013;79:616e24. [11] Rebordao MR, Galvao A, Pinto-Bravo P, Pinheiro J, Gamboa S, Silva E, et al. Endometrial prostaglandin synthases, ovarian steroids, and oxytocin receptors in mares with oxytocin-induced luteal maintenance. Theriogenology 2017;87:193e204. [12] Vanderwall DK, Parkinson KC, Rigas J. How to use oxytocin treatment to prolong corpus luteum function for suppressing estrus in mares. J Equine Vet Sci 2016;36:1e4. [13] Ginther OJ. Prolonged luteal activity in mares e a semantic quagmire. Equine Vet J 1990;22:152e6. [14] Gee E, Gillespie L, Bolwell C. Effect of oxytocin on suppression of oestrus in mares exhibiting normal oestrous cycles. N Z Vet J 2012;60:189e93.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Using Estrous Behavior to Time Initiation of Oxytocin Administration to Prolong Luteal Function in Mares Hannah S. Manning, Erin E. Runcan*, Christa R. Dias de Moraes, Marco A. Coutinho da Silva Department of Veterinary Clinical Sciences, The Ohio State University College of Veterinary Medicine, Columbus, OH
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2018 Received in revised form 29 January 2019 Accepted 30 January 2019 Available online 6 February 2019
The objective of this study was to use estrous behavior alone to determine the appropriate time for beginning an oxytocin treatment protocol for estrus suppression. We hypothesized that administration of oxytocin beginning 8 days after the onset of estrus will prolong the luteal phase in mares. Twentythree light breed mares (aged 4e20 years) were exposed to a stallion and observed for signs of sexual receptivity. Mares not displaying signs received 250 mg of cloprostenol intramuscularly (IM) and were teased again 3e4 days later. On the day that estrous behavior was observed (Day 0), mares were randomly divided into two groups: oxytocin (n ¼ 11): oxytocin (60 IU, IM) was administered once daily from Day 8e17; control (n ¼ 12): did not receive treatment. Blood was collected from all mares every 4 days throughout Day 17, and every 7 days thereafter until Day 45. Serum progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. Interestrus interval was defined as the period between Day 0 and the day when progesterone next reached <1.0 ng/mL. The average interestrus interval was higher for treated mares compared with control mares (32.4 ± 4.2 vs. 21.8 ± 1.5 days, respectively, P ¼ .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 (54.5%) mares and up to 45 days in 5 of 11 mares (45.5%). We conclude that luteal maintenance beyond 30 days was attained by once-daily oxytocin administration beginning 8 days following behavioral estrus in a majority of mares. © 2019 Elsevier Inc. All rights reserved.
Keywords: Horse Oxytocin Mare Estrus Behavior Corpus luteum
1. Introduction Poor performance due to undesirable estrous behavior is a common complaint made by owners and trainers of performance mares. Typically, the mare is in a period of estrus or sexual receptivity toward the stallion from 5 to 7 days during the estrous cycle. Such estrous behaviors range from benign tail swishing, squealing, and frequent urination to more severe behavior such as poor attitude, aggressiveness, and difficulty handling and training [1]. Because these behaviors can be very problematic for performance horse owners and trainers, veterinarians are often consulted to determine ways to suppress estrus and eliminate the undesirable
Animal welfare/ethical statement: All animal use and procedures were approved by The Ohio State University, Institutional Animal Care and Use Committee, IACUC #2016A00000008. Conflict of interest statement: The authors declare no conflicts of interest. * Corresponding author at: Erin E. Runcan, Department of Veterinary Clinical Sciences, The Ohio State University College of Veterinary Medicine, 601 Vernon L. Tharp St., Columbus, OH 43210. E-mail address: [email protected] (E.E. Runcan). https://doi.org/10.1016/j.jevs.2019.01.012 0737-0806/© 2019 Elsevier Inc. All rights reserved.
behavior. Multiple methods may be used to suppress estrus including hormonal therapy with progesterone, intrauterine devices, and in extreme cases ovariectomy [1,2]. Hormonal suppression of estrus with progesterone has been a known, effective method of estrus suppression for many years, and there are many different formulations. Natural progesterone in an oil base can be administered intramuscularly once daily. However, this method of administration can result in local tissue inflammation and pain as a result of the repeated injections. It is therefore not commonly used for suppression of estrus in performance mares [3]. Long-acting progesterone preparations have also been reported to be efficacious for estrus suppression. This preparation is administered once via an injection intramuscularly. There are 12day and 30-day formulations available; however, these longacting preparations may not be as effective as once-daily treatment and also have the ability to cause muscle soreness on injection [1]. Synthetic progesterone products are another method of estrus suppression. Altrenogest is the only highly effective and safe progestin approved for use in mares. It is dosed orally and administered once daily. Although very effective, treatment must be administered 3e4 days before an event and continued daily
H.S. Manning et al. / Journal of Equine Veterinary Science 75 (2019) 78e81
throughout the showing period [1]. Owners and trainers administering altrenogest are also at an increased risk, as it can have adverse health effects with inappropriate human exposure [4]. Veterinarians have also suppressed estrus in mares with the use of an intrauterine glass marble. Studies have shown this method is inexpensive and easy to administer; however, the efficacy is limited (41.7%) [5]. There have also been many risks reported in association with the use of glass marbles. For example, the prolonged presence of a glass marble has been associated with pyometra, chronic intermittent colic, and even the establishment of pregnancy and subsequent abortion. There is also a potential for harm to the mare, should the glass marble be damaged during removal or while inside of the uterus [6]. Finally, for extreme cases, ovariectomy is another method used to suppress estrus. This method would ultimately remove the source of the hormones producing aberrant estrous behavior. However, this method is considered a last resort, as it is permanent and irreversible [1]. Surgical complications, although uncommon, are possible and include hemorrhage, infection, adhesions, herniation, and evisceration. The procedure is also not 100% effective, with a small percentage (20%) of mares continuing to exhibit estrus-like behaviors postovariectomy in one study [7]. Previous research has shown that administration of exogenous oxytocin from Days 7e14, during diestrus, is a very effective, lowcost, and practical method of suppressing estrus [8,9]. The protocol works by inhibiting luteolysis, extending function of the corpus luteum, and maintaining elevated levels of the steroid hormone progesterone in the bloodstream, which suppresses behavioral estrus. The mechanism of prolonged function of the corpus luteum via exogenous oxytocin administration before Day 10 has been shown to be a result of oxytocin preventing the upregulation of cyclooxygenase-2 expression in the endometrium causing subsequent impairment of PGF2a secretion and synthesis [10,11]. Vanderwall et al reported luteostasis in 100% of horses treated in this initial “proof of principle study” [8]. The researchers then went on to report 60%e70% efficacy in prolonging the corpus luteum when evaluating once-daily versus twice daily-oxytocin administration in a larger population of mares [12]. Although exogenous oxytocin administration to suppress estrus is successful, it currently requires serial costly veterinary visits to determine the exact date of ovulation. The use of transrectal ultrasonography and palpation to detect ovulation and estrus in the mare must occur before the protocol can begin. Thus, the present study aims to eliminate such veterinary visits, as it may not be feasible in all situations. The objective of this study was to determine whether oxytocin administration beginning at Day 8 after behavioral onset of estrus will be effective in prolonging the luteal phase in mares, without knowing the exact date of ovulation.
79
signs used to indicate sexual receptivity included posturing toward the stallion, squatting to urinate, winking, and so on. When mares were teased, they were designated positive, passive, or negative. If mares did not show any signs of sexual receptivity (squealing, kicking, pinning of the ears, tail clamping, or actively moving away from the stallion), they were considered negative. Luteolysis was induced (250 mg Cloprostenol, intramuscularly (IM); Merck Animal Health, Madison, NJ) for these mares, and teasing was resumed 3e4 days later. If mares teased passively (standing quietly, allowing the stallion to touch without kicking, ears neutral), they were continued to be teased once daily until they showed more positive indicators of estrus. On the first day mares teased positive or exhibited behavioral estrus (Day 0), mares were randomly assigned to an experimental group (n ¼ 11) or control group (n ¼ 12). The control group did not receive treatment and was used to confirm a normal estrous cycle and progesterone profile. In the experimental group, mares received 60 IU of Oxytocin IM (right or left neck; Bimeda e MTC Animal Health, Cambridge, Ontario, Canada) once a day starting on Day 8 and continuing until Day 17. Treatment was initiated on Day 8 to account for a normal estrus period of 5e7 days and allow sufficient time for ovulation to occur. Beginning on Day 0, jugular blood samples (10 mL) were drawn via a vacutainer from experimental and control groups every 4 days through Day 17 and every 7 days thereafter until Day 45. The experiment was conducted through Day 45 to allow time to demonstrate efficacy of the protocol beyond the duration of a normal physiological estrous cycle and still remain within the time constraints of the study. 2.3. Progesterone Assay Blood samples were allowed to coagulate and then centrifuged at 2000 g for 10 minutes. The serum was drawn off of the samples and frozen at 80 C until analysis. Samples were run in house at The Ohio State University Veterinary Hospital using a Siemens/DPC Immulite Analyzer (The Ohio State University, Columbus, OH) to determine progesterone concentration. Progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. 2.4. Statistical Analysis
2. Materials and Methods
The interestrus interval was defined as the period between Day 0 and the day when serum progesterone next reached <1.0 ng/mL. The average interestrus interval between groups was compared by independent samples t-test using Excel's statistical software package. Data are presented as mean ± standard error of the mean. Significance was set at P < .05. The proportion of extended interestrus intervals between groups was compared using Fisher's Exact Test.
2.1. Animals
3. Results
This study was conducted in the northern hemisphere between the months of May and October. Twenty-three light breed mares, 4e20 years of age and owned by The Ohio State University College of Veterinary Medicine for teaching and research, were used for this study. All animal procedures were approved and conducted according to the guidelines of The Ohio State University Institutional Animal Care and Use Committee.
The average interestrus interval was higher for oxytocin treated mares compared with the control group, 32.4 ± 4.2 versus 21.8 ± 1.5 days, respectively (P ¼ .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 mares (54.5%) and up to 45 days in 5 of 11 mares (45.45%; Figs. 1A and 1B). Progesterone levels oscillated normally in 11 of 12 control mares. Two control mares were enrolled in the study but were not truly in estrus, made evident by progesterone concentrations >1.0 ng/mL at Day 0. One control mare experienced a spontaneously prolonged luteal phase of her cycle, which is evident by persistently elevated progesterone beyond 25 days (Fig. 2). When comparing 1 of 12 control
2.2. Experiment Twenty-three light breed mares were teased to a stallion and observed for signs of sexual receptivity or behavioral estrus. The
H.S. Manning et al. / Journal of Equine Veterinary Science 75 (2019) 78e81
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Fig. 1. (A) Blood progesterone levels from mares treated with oxytocin and resultant prolongation of luteal function. The red line indicates the progesterone reference (1.0 ng/mL) used to determine whether mares had a functioning corpus luteum (>1.0 ng/mL indicated a functioning CL). Day 0 was the first day mares displayed behavioral signs of estrus. (B) Blood progesterone levels from mares treated with oxytocin that did not result in prolongation of luteal function. The red line indicates the progesterone reference (1.0 ng/mL) used to determine whether mares had a functioning corpus luteum (>1.0 ng/mL indicated a functioning CL). Day 0 was the first day mares displayed behavioral signs of estrus.
4. Discussion
mares with a naturally extended interestrus interval with 6 of 11 experimental mares with an extended interestrus interval, the onetailed P value equaled .02. The comparison between 1 of 12 control mares and 5 of 11 experimental mares that had an extended luteal phase beyond 45 days yielded a one-tailed P value of .06.
These results confirm that administration of exogenous oxytocin on Days 8e17 post behavioral onset of estrus was efficacious in 54.5% of mares. To ensure treatment was based on behavior alone,
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Time (Day of the Cycle)
Fig. 2. Blood progesterone levels from mares that did not receive oxytocin treatment and served as controls. The gray line represents the mare with suspected retained corpus luteum. The red line indicates the progesterone reference (1.0 ng/mL) used to determine whether mares had a functioning corpus luteum (>1.0 ng/mL indicated a functioning CL). Day 0 was the first day mares displayed behavioral signs of estrus.
H.S. Manning et al. / Journal of Equine Veterinary Science 75 (2019) 78e81
transrectal ultrasonographic evaluation was not performed to document ovulation. As described in previous work by Vanderwall et al [12], efficacy of this treatment protocol would be improved by documenting that ovulation had indeed occurred rather than waiting a set time of 8 days from the appearance of behavioral estrus. When comparing the extended interestrus intervals to 30 days, there was a significant difference between the one control mare and six experimental mares that had a prolonged luteal phase. When evaluating the group that had an extended luteal phase beyond 45 days, 1 of 12 control mares compared with 5 of 11 experimental mares was not statistically significant (P ¼ .06); however, there was clearly a positive trend noted in this group of mares. This result would have likely been significant had a larger treatment group been evaluated. Because treatment enrollment was based on estrous behavior alone, variability exists as to the exact day in estrus that defines “Day 0.” Because of this, it is not surprising that some mares did not adequately respond. In previous work by Rebordao et al [11], it has been noted that oxytocin is antiluteolytic when treatment is initiated before Day 10 of diestrus and proluteolytic when administered after Day 14. In our subset of mares that did not respond to treatment, it is apparent that the initial injections of oxytocin began too early in diestrus to be effective, which is evident by the lack of elevated progesterone at the time of oxytocin administration. Our results are lower than reported success rates obtained in previous studies that initiated oxytocin treatments based on the day of ovulation (60%e70%, 100%) [8,12]. The same authors reported improved efficacy when oxytocin treatment duration was extended up to 29 days [12]. Keith et al also reported increased efficacy of oxytocin treatment with increasing number of treatment days (Days 8e10 vs. Days 8e12 vs. Days 8e14 postovulation) [10]. Potentially, the success rate of the present study would be improved if oxytocin treatment was initiated further into diestrus, and the duration of treatment was extended beyond 10 days. This would provide a longer duration of treatment to ensure continuation through the end of diestrus, especially because the date of ovulation is unknown. For the control group (Fig. 2), 11 of the 12 mares had a normal estrous cycle and progesterone profile. A subset of control mares depicted in Fig. 2 was only evaluated for 31 days because of time constraints where they were housed. We suspect the mare with elevated progesterone (>1.0 ng/mL) throughout her cycle had a prolonged luteal phase either from a retained corpus luteum or a diestrus ovulation. The phenomena of a persistent luteal phase are relatively common in the mare, occurring in up to 25% of documented equine estrous cycles [13]. The exact reason for the persistently elevated progesterone in this control mare is unknown, as ovarian structures were not monitored through the study. In our study, we used progesterone concentrations alone to determine the persistence of the luteal phase in experimental mares, indicated by serum progesterone above 1.0 ng/mL. Although estrous behavior was not assessed throughout the study, it is expected that mares with progesterone concentrations above 1.0 ng/ mL would not show estrous behavior. Based on serum progesterone concentrations alone, mares with progesterone concentrations above 1.0 ng/mL should have an active corpus luteum and should therefore not show estrous behavior [14]; however, it is important to note that not all mares show the same behavioral indicators of estrus. Some mares showed fairly subtle signs, making the use of
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behavior alone for determining when to initiate oxytocin treatment challenging. We suspect this is why two of the control mares were enrolled in the protocol that were not truly in estrus as depicted by progesterone concentrations >1.0 ng/mL on Day 0. 5. Conclusion Luteal maintenance beyond 31 days was attained in 54.5% of mares by once-daily oxytocin administration beginning 8 days following behavioral signs of estrus. The outcomes of this study are relevant to cases in which multiple veterinary visits (including transrectal palpation and ultrasonography) may not be feasible in all situations. One major limitation of this protocol is the requirement that either a stallion be present on farm to evaluate behavior or a veterinary examination be performed to confirm the mare is in fact in estrus at the time the aberrant behavior is noted. Further research is needed to improve efficacy of this protocol. We believe that efficacy may be improved by beginning treatment later into diestrus (beyond Day 8) and increasing the treatment duration. With modifications and further research, we believe this protocol could be a practical and low-cost method of estrus suppression for performance horse owners and trainers. Financial Disclosure This work was supported by The Ohio State University Veterinary Scholars Summer Research Program. References [1] McCue PM. Estrus suppression in performance horses. J Equine Vet Sci 2003;23:342e4. [2] McCue PM. Hormone therapy in clinical equine practice. Vet Clin North Am Equine Pract 2016;32:425e34. [3] Loy RG, Swan SM. Effects of exogenous progestogens on reproductive phenomena in mares. J Anim Sci 1966;25:821e6. [4] Fact Sheet, Regu-Mate®. Intervet Inc., Merck Animal Health; 2012. https:// www.merck-animal-health-usa.com/pdfs/equine/Regu-Mate%20-Fact%20Sheet %20-%20Merck%20Animal%20Health.pdf. [Accessed 15 February 2019]. [5] Nie GJ, Johnson KE, Braden TD, Wenzel JGW. Use of an intra-uterine glass ball protocol to extend luteal function in mares. J Equine Vet Sci 2003;23:266e73. [6] Vanderwall DK. Is it time to retire the use of intrauterine glass balls for estrus suppression in mares? J Am Vet Med Assoc 2015;247:346e7. [7] Roessner HA, Kurtz KA, Caron JP. Laparoscopic ovariectomy diminishes estrusassociated behavioral problems in mares. J Equine Vet Sci 2015;35:250e3. [8] Vanderwall DK, Rasmussen DM, Woods GL. Effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares. J Am Vet Med Assoc 2007;231:1864e7. [9] Vanderwall DK, Rasmussen DM, Carnahan KG, Davis TL. Effect of administration of oxytocin during diestrus on corpus luteum function and endometrial oxytocin receptor concentration in cycling mares. J Equine Vet Sci 2012;32:536e41. [10] Keith L, Ball BA, Scoggin K, Esteller-Vico A, Woodward ET, Troedsson MHT, et al. Diestrus administration of oxytocin prolongs luteal maintenance and reduces plasma PGFM concentrations and endometrial COX-2 expression in mares. Theriogenology 2013;79:616e24. [11] Rebordao MR, Galvao A, Pinto-Bravo P, Pinheiro J, Gamboa S, Silva E, et al. Endometrial prostaglandin synthases, ovarian steroids, and oxytocin receptors in mares with oxytocin-induced luteal maintenance. Theriogenology 2017;87:193e204. [12] Vanderwall DK, Parkinson KC, Rigas J. How to use oxytocin treatment to prolong corpus luteum function for suppressing estrus in mares. J Equine Vet Sci 2016;36:1e4. [13] Ginther OJ. Prolonged luteal activity in mares e a semantic quagmire. Equine Vet J 1990;22:152e6. [14] Gee E, Gillespie L, Bolwell C. Effect of oxytocin on suppression of oestrus in mares exhibiting normal oestrous cycles. N Z Vet J 2012;60:189e93.