[Descriptive comparison of serum progesterone levels during intravaginal device insertion in mares]

carried out with a larger number of animals

The equine industry plays an important role in the national and international agribusiness and the valuation of time savings and human resources is increasingly relevant in the field routine.Therefore, the use of hormonal protocols associating progesterone (P4) and estradiol (E2) to synchronize estrus and/or reduce the length of transitional periods are increasingly applicable in equine breeding systems.
In athletic mares, P4 is frequently used to reduce estrus behavior because, when associated with estradiol-17b, it plays a role of suppressing follicular development (Pinto et al., 2004).Additionally, P4-based protocols can promote estrus synchronization (Faria and Gradela, 2010;Fedorka et al., 2019) and cyclicity anticipation, decreasing the transition period and thus increasing the length of breeding season (Ginther et al., 2004;Satué and Gardón, 2013).


During P4 treatment, LH secretion is suppressed.Hence, the treatment with P4 inhibits estrus expression, interrupting the simultaneous follicular maturation and positively affecting the accumulation of LH in the pituitary.At the end of P4 treatment, a large secretion of LH is provided with formation of a dominant follicle with ovulatory capacity.Therefore, hormonal protocols with P4 supplementation can provide several favorable results when correctly applied and outside the period of deep anestrus (Handler *Corresponding author: leticiazoccolaro@yahoo.com.br;leticiazo@vet.ufmg.brSubmitted: March 22, 2023. Accepted: June 2, 2023. et al., 2006;Hanlon and Firth, 2012;Teixeira et al., 2021).
Progestogens are synthetic compounds that act on P4 receptors and may belong to different molecular classes and be available in different ways (Piette, 2018).In general, oral and injectable synthetic progestins are commercially available, as well as intravaginal devices (Faria and Gradela, 2010).Intravaginal progestogens are advantageous because they are easily applied and do not require daily treatment and/or injections (Negretti et al., 2018).
In cattle, intravaginal P4 devices are widely used and ensure ovulation blockade, even when the implant is reused (Oliveira et al., 2019).This second use depends on the initial P4 concentration of the device and animal category, but it is presented as an alternative to reduce the cost of the protocol.However, in mares, no studies were found in the literature using seconduse devices or describing the serum concentrations of P4 during the device insertion.Therefore, the present study aims to describe the serum levels of P4 in mares treated with one or two first-use (new) intravaginal devices and even with a second use (reused) device in a period of ten consecutive days and to compare the P4 concentration obtained throughout the experimental period of the three treatments.The experiment was performed on an experimental farm located in Esmeraldas (tropical climate), in the state of Minas Gerais,Brazil (latitude 19°45'46'' S,longitude 44°18'47'' W).Females were kept on the same pasture of Cynodon dactylon with water ad libitum, during the month of September 2021 (CEUA UFMG 49/2021).

Arq
Three crossbred healthy mares, aged between 8 and 14 years and body condition score 3 (Carroll and Huntington, 1988) were selected for the study.A previous gynecological examination was performed for each mare to assure the absence of corpus luteum in the ovaries and the absence of vaginal discharge, endometrial edema, or uterine alterations.
The intravaginal P4 release device used in this study was the Reprosync (GlobalGen, São Paulo, Brazil) containing 2.0 g of progesterone.In all animals the P4 devices were maintained for 10 days after insertion (D0 to D10).Mare 1 received two new (first use) P4 devices (two devices containing 2g of P4 each), mare 2 received a new P4 device (one device containing 2g of P4) and mare 3 received reused (second use) P4 device (a 2g P4 device previously used in another adult mare, for 10 days, before the experiment).
Blood collections for serum P4 dosage were performed before insertion of the device (D0), and daily, during the 10 days the device was in use (D0 to D10), in the morning (9:00am) and in the period of afternoon (15:00 pm).
In the case of the used implant (second-use implant), after it was used for the first time, it was washed in a bucket with water and soap to remove dirtiness (after its removal from the first use) and then it was washed with running water.Subsequently, the device was cleaned with a 1:3000 CB30 TA® solution (Alkyl dimethyl benzyl ammonium chloride, Ourofino Saúde Animal, São Paulo, Brazil) for 5 minutes.After drying in the shade and at room temperature, it was stored in its original packaging until reuse.
Blood samples were obtained using siliconized tubes (red cap) and a 25mm x 8mm needle (Vacutainer® system), reserved at room temperature for 1 hour to clot.Then, the tubes were centrifuged (FANEM, model excelsa baby) for 15 minutes at 900xg at 20°C.The aliquoted serum was stored in polystyrene tubes at -20°C until analyzed.
Progesterone concentrations were analyzed by using the chemiluminescence method (IMMULITE 2000 XPi immunoassay system; Siemens Healthineers, Erlangen, Germany, USA) as described by Fazio et al.
In the present work, serum P4 concentrations of mares receiving different amounts of intravaginal P4 were described.The intravaginal P4 devices have been used in horses because it is a noninvasive and practical procedure that allows rapid and prolonged absorption of P4 (Pohl et al., 2009;Negretti et al., 2018).Nevertheless, the commercially available devices were designed for the bovine anatomy which may cause losses of these devices when used in mares, or even undesirable local inflammatory reactions (Polasek et al., 2017.In the present study, however, the P4-releasing intravaginal device used (commercially indicated for cattle) was easy to apply in the mares and no occurrences of expulsion or losses were observed during the days of the experiment.Additionally, in the mares of the present study, no vaginal discharge was identified.
Hence, considering that the use of intravaginal devices can lead to inflammatory process of vaginal mucosa due to the mechanical action they exert in the organ (Faria and Gradela, 2010;Polasek et al., 2017, the importance of impregnating the devices with antibiotics prior to insertion is highlighted, as well as the correct hygiene of the second-use device, to minimize the chances of developing vaginitis (Rutten et al., 1986).In the study of Handler et al. (2006), which evaluated the use of intravaginal devices in mares, no previous use of antibiotics was performed and approximately one third of the animals presented moderate vaginitis.Still, according to other authors, when vaginitis occurs it is spontaneously resolved and does not have a negative impact on the total number of follicles, follicle diameter, ovulation rates, interval between P4 removal and ovulation, endometrial edema, or pregnancy rate, and does not affect embryo losses in recipient mares (Polasek et al., 2017;Segabinazzi et al., 2021).
In the present work, at the time that devices were inserted, none of the three mares had a corpus luteum (CL), which was confirmed by the serum hormone dosage showing P4 values below 1 ng/ml on D0 (am), as demonstrated in Table 1.From the insertion of intravaginal devices, serum P4 concentrations increased in all mares of the study and the values were already above 1ng/mL at six hours after the insertion (Table 1).
As previously demonstrated, regardless of seasonality, transvaginal absorption of P4 occurs immediately and effectively in the first hours after insertion (Handler et al., 2006;Pohl et al., 2009).The high absorptive capacity of vaginal mucosa and the type of material used for production of intravaginal implants allows this rapid hormone level rise (Handler et al., 2006;Segabinazzi et al., 2021).
The release mechanism of P4 from the device occurs by passive diffusion, and the hormone transfer is directly proportional to the magnitude of the concentration gradient across the membrane, the P4 liposolubility coefficient and the exposed surface area.In addition to the rapid hormone absorption right after the device insertion, this mechanism enables slow hormone release, maintenance of plasma levels for long periods (during the period of device presence) and sharp drop in P4 serum concentrations right after the device removal (Lavy et al., 2006;Brunton and Parker, 2008).hours after P4 insertion (at 15:00 of day 0); D2 (am): P4 concentration assessed in the morning of the second day of device presence (at 09:00 am of day 2); D2 (pm): P4 concentration assessed in the afternoon of the second day of device presence (at 15:00 of day 2); D4 (fourth day of device presence); D6 (sixth day of device presence); D8 (eighth day of device presence); D10 (am): P4 concentration assessed in the morning of the tenth day (last day) of the device insertion (at 09:00 am of day 10); D10 (pm): P4 concentration assessed 6 hours after P4 device removal (at 15:00 of day 10).
In Table 1 we also observe a sudden drop in the serum concentrations of the hormone in the 3 mares.However, in the mare that received two P4 devices (mare 1), the decrease in P4 concentrations occurred more slowly.At six hours after device removal, this animal still presented values above 1ng/mL due to the need for more time to metabolize the higher concentration of circulating P4 observed throughout the experimental period in this animal.
Similarly, Bisinotto et al. (2015) showed, in dairy cows, that after the device removal the serum P4 concentrations using two intravaginal devices decreases more slowly compared to one device (with values greater than 1ng/ml) and that the P4 levels of animals with two devices are closer to the hormone values produced by the CL compared to only one device.Also in dairy cows, Vasconcelos et al. (2018) showed that the use of a second device had a positive influence on follicular growth and oocyte quality compared to Arq.Bras.Med.Vet.Zootec., v.75, n.5, p.1019-1025, 2023 animals with one device.Additionally, an improvement in the fertility of dairy cows was confirmed in the animals that ovulated larger follicles due to the increased circulating P4 promoted by the doble device (Vasconcelos et al., 2018).
Considering the mean P4 levels throughout the entire experimental period for each animal (Table 2), it was observed that the mare that received two P4 implants (mare 1) had numerically higher mean serum P4 concentration than the mare that received one new P4 device or than the mare that received one used P4 device, which may be related to the larger contact surface of the two implants, compared with the contact surface with the vaginal mucosa of only one implant.Silva et al. (2021), using nonlactating Holstein cows, demonstrated that the size of surface area affects the ability of devices to release P4 since the greater the contact surface area, the greater the circulating concentrations of P4.Because the vaginal epithelium is highly permeable to steroids (and analogues), it is considered a fast and effective route of absorption for releasing the hormone into the circulation (Villanueva et al., 1981;Rothen-Weinhold et al., 2000;Corleta et al., 2004).
On the other hand, it was interesting to note that the mares receiving one new device (mare 2), or a previously used device (mare 3), had relatively similar means of P4 levels, numerically, over the experimental period (Table 2).To the authors' knowledge, this is the first report to describe serum P4 concentrations in mares with a seconduse intravaginal implant and it is also the first report to demonstrate that, in mares, the mean serum P4 concentrations seems to be similar in animals with new or reused intravaginal devices.
On this basis, it may perhaps be speculated that, after the first use in mares, the levels of P4 impregnated in the device remained high enough to be reused in mares, and/or that the surfaces area in contact with the vaginal mucosa of both mares have the same limits of P4 absorption (by the vaginal mucosa), which prevents the animal that received a new implant from having more circulating P4.   et al. (1976), a minimum circulating concentration of 2 ng/ml of P4 is needed so that ovulation does not occur during the period of permanence of P4 devices in mares.Because P4 exerts a negative feedback effect on the hypothalamic-pituitary axis and blockage of the cyclic LH release from the anterior pituitary, it suppresses estrus behavior of mares if plasma P4 concentrations exceed 1 to 2ng/mL (Aurich and Kaps, 2022).During the whole period of device permanence, serum P4 levels lower than 1 ng/ml were not observed in any of the three mares.Therefore, all treatments used in the present study (two new implants, one new implant or one used implant) seem to be possible options to prevent mares from ovulating while remaining intravaginally inserted, up to 10 days in the animals, although further studies (with a larger number of animals) are needed to confirm these findings.

Figure 1 .
Figure 1.Serum progesterone (P4) concentration in mares with intravaginal P4 device; D0-0H: Serum progesterone (P4) concentration before device insertion; D0-12H: Serum P4 concentration 12 hours after P4 insertion; D1: serum P4 concentration at the first day (day 1) of device presence; D2 (pm): serum P4 concentration at the second day (day 2) of device presence; D10-AM: serum P4 concentration assessed during the morning of the tenth day (day 10) of the device insertion; D10-PM: P4 concentration assessed 12 hours after P4 device removal.It is also noted in Fig.1that serum P4 levels remained relatively similar in mares that received one implant (new or used) throughout the experimental period.Additionally, until the end of P4 treatment (D10-AM), the serum P4 concentrations reached values above the minimum necessary to avoid ovulation, in the three mares of the experiment.According toNett  et al. (1976), a minimum circulating concentration of 2 ng/ml of P4 is needed so that ovulation does not occur during the period of permanence of P4 devices in mares.Because P4 exerts a negative feedback effect on the hypothalamic-pituitary axis and blockage of the cyclic LH release from the anterior pituitary, it suppresses estrus behavior of mares if plasma P4 concentrations exceed 1 to 2ng/mL(Aurich and Kaps, 2022).During the whole period of device permanence, serum P4 levels lower than 1 ng/ml

Table 1 .
Descriptive values of serum progesterone (P4; ng/mL) concentration of mares receiving two new