Comparison of two methods of synchronization of estrus in brown brocket deer (Mazama gouazoubira)

Animal Reproduction Science 117 (2010) 266–274

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Comparison of two methods of synchronization of estrus in brown brocket deer (Mazama gouazoubira) Eveline dos Santos Zanetti a,b,∗ , Bruna Furlan Polegato a , José Maurício Barbanti Duarte a a

Deer Research and Conservation Center (NUPECCE – Núcleo de Pesquisa e Conservac¸ão de Cervídeos), Departamento de Zootecnia, 14884-900 Jaboticabal, SP, Brazil b Programa de Pós-graduac¸ão em Medicina Veterinária, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, 14884-900 Jaboticabal, SP, Brazil

a r t i c l e

i n f o

Article history: Received 13 January 2009 Received in revised form 2 May 2009 Accepted 11 May 2009 Available online 18 May 2009 Keywords: Estrus Mazama gouazoubira Fecal progestins Fecal estrogens Brown brocket deer

a b s t r a c t This study aimed to establish a protocol for synchronization of estrus in brown brocket deer (Mazama gouazoubira). Two groups of hinds (n = 3) were submitted to two different protocols: Treatment 1 received an intravaginal progesterone (CIDR® ) device for 8 days, followed by 265 ␮g injection of cloprostenol at the time of removal; and Treatment 2 received two injections of 265 ␮g of cloprostenol 11 days apart. After 30 days, each group of three hinds received the other treatment. Treatment efficacy was evaluated by reproductive behavior, fecal progestin and estrogen concentration and the observation of CL by laparoscopy 6 days after the end of estrus. All the hinds (100%) had estrous behavior upon the completion of treatment, but a significant difference occurred between the time of onset, 70.5 ± 5.0 h for Treatment 1 and 52.3 ± 5.6 h for Treatment 2. The mean estrus duration time (34.7 ± 4.50 and 37.0 ± 8.11 h), ovulation rates (5/6 and 4/6), mean CL size (4.85 ± 0.74 and 3.21 ± 0.19 mm) and mean fecal progestin concentration at 6 days after the end of estrus (865.53 ± 76.59 and 1073.35 ± 106.82 ng/g feces) were not significantly different between treatments. There was no difference in fecal estrogen concentrations throughout the treatment and the greatest values of the estrogen:progestin ratio coincided with estrous behavior. Although fertility was not evaluated directly, both treatments were effective in synchronizing estrus in the species M. gouazoubira, with the formation of functional corpora lutea. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The Neotropical region is passing through a critical period in terms of loss of biodiversity. The accentuated reduction in flora and fauna populations is related to this process, leading to the loss of heterozygosity, increased endogamy and, consequently, greater susceptibility to environmental changes and greater risk of extinction (Duarte, 2005). In this scenario, of the 17 species of Neotropical deer species, 59% are classified as vulnerable to or threat-

∗ Corresponding author. Tel.: +55 16 3209 2678. E-mail address: eveline [email protected] (E.S. Zanetti). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.05.010

ened with extinction (IUCN, 2008). However, reproductive technologies offer new solutions to facilitate the genetic management of endangered species, such as the development of genetic resource banks (Holt and Pickard, 1999; Wildt and Wemmer, 1999; Roldan et al., 2006). Accurate and predictable detection of estrus and ovulation is essential for the success of assisted reproduction, particularly for artificial insemination (AI) and the collection and transfer of embryos (ET) (Asher et al., 1992). These methods are more reliable than the detection of natural estrus and permit the precise timing of the onset of estrus and ovulation (Hodges, 1996). Synchronization of estrus is typically achieved by the administration of progestogens, the injection of prostaglandins and their synthetic

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analogues (cloprostenol, for instance) or a combination of both (Hosack et al., 1999; Morrow et al., 2000). For the family Cervidae, almost all detailed reproductive knowledge concerning synchronization of estrus have been derived from studies of temperate species that exhibit seasonal cycles (Cervus elaphus—Asher et al., 1992, 1993, 1995; Fisher et al., 1994; Fletcher, 2001; McCorkell et al., 2007; Dama dama—Asher and Smith, 1987; Asher and Thompson, 1989; Asher et al., 1993; Jabbour et al., 1993a; Morrow et al., 1995; Odocoileus virginianus—Magyar et al., 1989) that can therefore be submitted to induced estrus. However, to our knowledge, information regarding synchronization of estrus in deer species native to South America is limited. Among Brazilian species, synchronization of estrus has only been described in the species Mazama gouazoubira, using home-made sponges of medroxyprogesterone, for 14 days, and cloprostenol, two injections 10 days apart (Duarte and Garcia, 1995). The brown brocket deer (M. gouazoubira) is one of the most abundant deer species in the Neotropical region (Duarte, 1996) and is considered a nonseasonal breeder, giving birth at all times of the year (Pereira et al., 2006). Data from behavioral studies indicate the hinds are polyestrous, with a mean estrous cycle duration of 26.9 ± 1.7 days (range of 21–37 days), and a mean length of behavioral estrus of 2.3 ± 0.2 days (range of 1–4 days). Based on progestogen data, the estrous cycles consisted of a 24.6 ± 1.4 day luteal phase and a 1.7 ± 0.1 day inter-luteal phase (Pereira et al., 2006). These studies have generated a useful database, because basic knowledge of the reproductive physiology of a species is an essential factor for initiating the development of reproduction biotechniques (Comizzoli et al., 2000). For this reason, the brown brocket deer has been used as an experimental model to evaluate reproduction techniques as a tool for Neotropical deer conservation. The aim of the present study was to establish a protocol for stage of estrous cycle synchronization in brown brocket deer (M. gouazoubira), evaluate the onset and duration of estrus, the ovulation rate and the presence and functionality of corpora lutea after synchronization by two different methods. 2. Materials and methods The present study was approved by the Animal Ethics and Welfare Committee (Comitê de Ética e Bem-estar Animal, CEBEA) of the Faculty of Agrarian and Veterinary Sciences (Faculdade de Ciências Agrárias e Veterinárias, FCAV) UNESP, Jaboticabal, SP, Brazil. 2.1. Animals Six adult hinds (four primiparous and two nulliparous, aged 2.3–9.0 years old, weighing 15.3–19.9 kg) and 1 male (vasectomized, 6 years old, weighing 17.5 kg) were housed at the Deer Research and Conservation Center (NUPECCE) facilities at the São Paulo State University (UNESP)/Jaboticabal Campus (20◦ S latitude). From March through July 2005, all the deer were maintained individually in stalls (4 m × 4 m) with auditory and olfactory contact with conspecific males and females and were exposed to

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normal fluctuations in photoperiod. They were fed ad libitum with a diet consisting of a pellet feed (12% crude protein, 2% crude fat, 10% crude fiber, Purina Co., Paulínia, São Paulo, Brazil) and approximately 1 kg/deer/day of fresh alfalfa (Medicago sativa), perennial soybean (Neonotonia wightii) or mulberry branches (Morus alba). Water was also provided ad libitum.

2.2. Treatments Hinds were allocated at random to one of two groups (n = 3) as follows: (i) Treatment 1, where the hinds each received a single intravaginal device impregnated with 0.33 g progesterone (CIDR-type T; Controlled Internal Drug Release, Pfizer, USA) for 8 days and an i.m. injection of a PGF2␣ analogue (265 ␮g cloprostenol; 1 mL Ciosin; Schering Plough Coopers, Brazil) upon removal of the device at 8 days; (ii) Treatment 2, where each hind was treated with two i.m. injections of a PGF2␣ analogue (265 ␮g cloprostenol; 1 mL Ciosin; Schering Plough Coopers, Brazil) 11 days apart. After 30 days, each group of three hinds received the other treatment. All treatment administrations were conducted under physical restraint (Duarte et al., 2001) between 11:00 and 14:00 h.

2.3. Detection of estrus Signs of estrous behavior were determined by allowing each hind to associate with a vasectomized male adult (5 min every 6 h) from 4 h after the completion of the treatments until the moment that the hind no longer accepted copulation (end of estrus). We defined behavioral estrus as the period in which hinds permitted copulation (Pereira et al., 2006).

2.4. Evaluation of the ovulation rate The ovulation rate was evaluated by CL counts 6 days after the end of estrus by laparoscopy, except for hind E (Treatment 1), which was evaluated by laparotomy. The procedure involved with-holding hinds from food and water for 24 h and physical restraint, followed by anesthesia with 5.0 mg/kg of ketamine hydrochloride (Dopalen; Vetbrans Saúde Animal, Jacareí, Brazil), 0.3 mg/kg of xylazine hydrochloride (Coopazine; Mallinckrodt Vet, Cotia, Brazil) and 0.5 mg/kg of medazolan (Dormonire; Cristália, Itapira, Brazil), by i.v. injection. To initiate the surgical process, the hinds were intubated and maintained on a superficial plane using isoflurane (Forane; Abbott, São Paulo, Brazil) at a mean concentration of 0.89 ± 0.08 V%, measured by a vaporizer calibrated for this drug (HB Hospitalar, São Paulo, Brazil). The laparoscopic procedures used were the same as described for red deer (Asher et al., 1992) and sheep (Godfrey et al., 1997). The incisions were closed with 2-0 nylon suture and the hinds were treated with benzathine penicillin (Pentabiotic; Fort Dodge, Brazil, 40,000 UI/kg i.m.) and phenylbutazone (Equipalazone; Marcolab, Brazil, 400 mg i.v.).

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2.5. Behavioral data and sample collection To identify the estrous cycle phase of the hinds, the reproductive cycles for each hind were monitored, from 30 days before the treatments to the end of the experiment, and signs of behavioral estrus were determined by allowing each hind to associate with a vasectomized male adult for 5 min daily, between 7:30 and 9:30 h. Fecal samples were collected daily between 7:30 and 9:30 h from the onset of treatment until 6 days after the end of estrus and on alternate days from 3 days prior to the onset of treatment. From the onset of behavioral estrus until 24 h after its completion, fecal samples were collected every 2 h. Each sample recovered within 12 h of voiding was placed in individually labeled plastic bags. The fecal samples were frozen (−20 ◦ C) within 20 min of collection. All samples were stored at −20 ◦ C until steroid analysis was performed. 2.6. Fecal steroid extraction and enzyme immunoassays The fecal samples (total amount collected at any given period) for each treatment were lyophilized for approximately 30 h (Model LGA05, Web LMW Medizintechnik, Leipizig, Germany). Dried fecal samples were pulverized and steroids were extracted from feces according to the method described by Graham et al. (2001). A proportion of the resulting powder (0.5 g) was weighed and extracted with 5 mL of 80% methanol. After vortexing for 30 s at high speed, the sample was shaken for 12 h on a mechanical shaker. After centrifugation at 1500 × g for 20 min, the supernatant was transferred into a clean tube. Aliquots of supernatant were diluted, 1:250 for the progesterone assay and 1:8 for the estradiol assay, for EIA analysis (Multiskan MS, Labsystem, Helsinki, Finland). The samples collected every 2 h were only assayed for estradiol, while all the other samples were assayed for progesterone and estradiol. The concentrations were determined using CL425 and R4972 antibodies (California University; Davis, CA, USA) for progestogens (P) and estrogens (E2), respectively. These antibodies were chosen due to their great amount of cross-reactivity with the metabolites excreted in M. gouazoubira feces—5␣- and 5␤-pregnanes and oestradiol-17␤ (Polegato, 2004). Validation of the hormone dosages was conducted according to Brown et al. (2004), by observation of the parallel disposition between the standard curve and that formed by the pool of fecal extracts prepared by serial dilution and by substantial recovery of exogenous progesterone (y = 0.99x + 1.14, r2 = 0.98) and estradiol-17␤ (y = 1.19x + 14.09, r2 = 0.89) added to fecal extracts. Interassay coefficients of variation for two separate controls were 12% (77 and 39% binding) for P and 25% (60% binding) and 15% (30% binding) for E2. Intra-assay coefficients of variation were <15%. All fecal data are expressed on a dry-weight basis. 2.7. Corpora lutea measurements The laparoscopy images were recorded on VHS, digitalized by the Amcap program for Windows as AVI files and later played on Windows Movie Maker. Three CL from each treatment were measured using the KS 300 Software (Kon-

tron Elektronik, Munich, Germany) by comparison with the tweezers used during the laparoscopy procedure. All measurements were determined by the mean of three consecutive verifications. 2.8. Statistical analysis Data analysis was performed using the Student’s t-test for independent measures and presented as the mean ± the standard error of mean (SEM). Nonparametric data (presence or absence of CL in the ovaries) were analyzed using the Fisher Exact test. The fecal hormone concentration values were submitted to analysis of variance following logarithmic transformation of hormone data (Morrow et al., 1995). For comparison of the means within each treatment, the Tukey test was used and for comparison of the means between treatments, the Student’s t-test was used. Hind D was excluded from the means for the inter-luteal phase and 6 days after the end of estrus due to repeated behavioral estrus after both treatments. Similar to the data analysis described by Thompson et al. (1998), baseline fecal progestin concentrations were defined for each hind separately by taking the least progestin value for each treatment; a mean and SD were calculated for these resulting six values. Values less than the criterion value (mean ± 2SD) were considered indicative of the inter-luteal phase. The mean fecal progestin concentration on day 6 after the end of behavioral estrus was the value used to compare the mean concentration of progesterone produced by the CL. The samples collected every 2 h and only assayed for estradiol were analyzed and expressed as the mean ± SEM per day. The E2:P ratio was calculated for the days on which both hormones were analyzed. All analyses were performed using the SAS program (SAS Institute Inc., Cary, NC, USA) and values of P < 0.05 were considered significant. 3. Results All the hinds (n = 6) expressed behavioral estrus after both treatments. A significant difference (P = 0.02) occurred between the times of behavioral estrus onset, but the same did not occur in relation to duration of estrus (Table 1). The time between the end of treatment and the onset of estrus was 52–88 h and 40–69 h for Treatments 1 and 2, respectively. Thus, synchrony, the interval between the first and last hind to exhibit estrous behavior, was shorter in Treatment 2 (Table 1). The duration of estrus was 24–52 h for Treatment 1 and 18–60 h for Treatment 2 (Table 1). Based on progestin data, Treatments 1 and 2 consisted of a 4.6 ± 1.57 day and 4.8 ± 1.39 day inter-luteal phase (P > 0.05), respectively, after removing hind D from the data set due to repeated behavioral estrus. Representative hormone response profiles of Female A with daily fecal progestin concentrations during Treatments 1 and 2 are shown in Figs. 1 and 2. Fecal progestin concentrations only decreased to inter-luteal levels 3.2 ± 0.73 and 2.0 ± 0.45 days after the completion of Treatments 1 and 2 (P > 0.05), respectively, such that, until 6 days after the end of behavioral estrus no significant increase in the mean daily fecal progestin concentrations occurred in the two treatments. Fecal progestin concentra-

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Table 1 Day of estrous cycle of each hind (day 0 = estrous behavior) from the onset of Treatments 1 (CIDR® for 8 days associated with cloprostenol on day 8) and 2 (two injections of cloprostenol 11 days apart) and their subsequent results in six hinds of the species Mazama gouazoubira. Treatment

Hinds

Cycle day

Time to onset of estrus (h)

1

A B C D E F

0 3 6 2 8 27

70 74 52 63 88 76

Mean ± SEM A B C D E F

2

Mean ± SEM a b c *

0 27 6 16 21 27

Synchronya (h)

Estrus duration (h)

Laparoscopy visualization

36

30 36 52 24 42 24

1 CL (lb ) 1 CL (rc ) + follicle (l) 1 CL (l) 1 follicle (r) 1 CL (l)* 1 CL (r)

70.5 ± 5.00a

34.7 ± 4.50a

40 69 40 62 63 40

42 18 60 18 60 24

52.3 ± 5.60b

29

1 CL (l) + follicle (l) 0 CL and 0follicle 1 CL (l) 1 follicle (l) 1 CL (l) 1 CL (r)

37.0 ± 8.11a

Interval between the first and last hind to exhibit estrus within each treatment. Left ovary. Right ovary. CL evaluation by laparotomy. Means within column with uncommon letters differ (P < 0.05) by the Student’s t-test.

Fig. 1. Excretion of fecal steroid hormone metabolites of one brown brocket deer (M. gouazoubira) hind (Female A) during Treatment 1; CIDR® associated with cloprostenol. Fecal progestin concentration (ng/g feces): full line; fecal estrogen:progestin ratio: empty line. Day zero = final day of treatment. Black bars indicate periods of estrous behavior.

Fig. 2. Excretion of fecal steroid hormone metabolites of one brown brocket deer (M. gouazoubira) hind (Female A) during Treatment 2; two applications of cloprostenol. Fecal progestin concentration (ng/g feces): full line; fecal estrogen:progestin ratio: empty line. Day zero = final day of treatment. Black bars indicate periods of estrous behavior.

270 Table 2 Fecal progestin concentrations [P4] and estrogen:progestin ratio (E2:P) of six hinds of the species M. gouazoubira, in the different phases of Treatments 1 (CIDR® for 8 days associated with cloprostenol on day 8) and 2 (two injections of cloprostenol 11 days apart). Hinds A B C D E F

1

Mean ± SEM A B C D E F

2

Mean ± SEM a

[P4] estrusa (ng/g feces)

[P4] inter-luteal phase (ng/g feces)

[P4] 6db (ng/g feces)

E2:P estrusc

E2:P inter-luteal phase

E2:P 6dd

938.66 899.84 377.81 391.89 316.98 505.37

476.06 409.54 323.10 – 383.78 426.57

1016.33 993.37 769.82 626.32 612.26 935.91

0.43 0.23 0.10 0.43 0.30 0.27

0.12 0.33 0.16 – 0.24 0.27

0.16 0.12 0.19 0.18 0.20 0.12

571.76 ± 112.77ac

403.81 ± 25.19ac*

865.54 ± 76.60ac*

0.29 ± 0.05ae

0.22 ± 0.04ae*

0.16 ± 0.02ae*

517.24 906.98 366.99 312.33 329.99 362.37

528.78 564.36 475.36 – 399.26 360.41

912.39 1190.14 1338.97 519.97 1178.99 746.27

0.15 0.20 0.36 0.45 0.60 0.24

0.15 0.26 0.23 – 0.56 0.26

0.03 0.09 0.14 0.35 0.15 0.08

465.98 ± 93.05ac

465.63 ± 38.28ac*

1073.35 ± 106.83ac*

0.33 ± 0.07ae

0.29 ± 0.07ae*

0.10 ± 0.02ae*

[P4] 1 day after the manifestation of behavioral estrus. [P4] on day 6 after the end of behavioral estrus. c E2:P 1 day after the manifestation of behavioral estrus. d E2:P on day 6 after the end of behavioral estrus. * Mean ± SEM excluding data from hind D, due to repeated behavioral estrus after both treatments. Means within column with uncommon letters (a and b) differ (P < 0.05) by the Student’s t-test. Means within row with uncommon letters (c and d) differ (P < 0.05) by the Student’s t-test. Means within row with uncommon letters (e and f) differ (P < 0.05) by the Student’s t-test. b

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Treatment

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tions in different phases of Treatments 1 and 2 are shown in Table 2. No differences occurred (P > 0.05) in fecal estrogen peak characteristics (mean peak concentration, day of peak, duration of elevated) or between the ratio of the concentrations of this hormone and progestins (Table 2). The greatest values observed for the E2:P ratio coincided with estrous behavior (Figs. 1 and 2). The ovulation rates evaluated by laparoscopy 6 days after the end of behavioral estrus were not significantly different between the two treatments (Table 1). The diameters of CL from Treatments 1 (4.85 ± 0.74 mm) and 2 (3.21 ± 0.19 mm) were also not significantly different. During the two laparoscopies of hind D, observation revealed the presence of large ovulatory follicles (3.75 and 5.58 mm in Treatments 1 and 2, respectively) and the absence of CL. This hind had repeated periods of behavioral estrus 1 day prior to and on the day of the first laparoscopy (Treatment 2). The same occurred 3 and 2 days prior to the second laparoscopy (Treatment 1). Hind B had no visible structures during Treatment 2 laparoscopy, despite presenting fecal progestin concentrations indicating the luteal phase (Tables 1 and 2). When CIDR® was removed, observation verified that 4 of 6 (66.67%) of the hinds presented hyperemia of the vaginal mucosa, with purulent secretion.

4. Discussion Both treatments (CIDR® for 8 days + cloprostenol on day 8 and 2 doses of cloprostenol 11 days apart) used in this research synchronized estrus in the species M. gouazoubira, as reported by Duarte and Garcia (1995), after the use of two doses of cloprostenol 10 days apart and the use of an intravaginal implant of medroxyprogesterone acetate for 14 days. However, the time between Treatment 1 completion and the onset of behavioral estrus was greater than the 48–72 h reported for the same species (Duarte and Garcia, 1995). The time the progesterone device remained in place in the latter experiment (14 days) was probably responsible for this difference. In general, the shorter the period of action of exogenous progesterone, the greater the range in the onset of estrus (Jabbour et al., 1991), as previously determined in the species D. dama (Morrow et al., 1995) and C. elaphus (Flint et al., 1997), for which signs of estrus occurred from 72 to 120 h, after 8 days of implantation, and from 81 to 96 h, after 12 days of implantation, respectively. Despite the concomitant use of cloprostenol, aimed to ensure that any persistent luteal tissue was removed at progesterone withdrawal, the short-term protocol presents a wide range in the onset of estrus, suggesting that this interval depends on the ovarian follicular development state at the time of progesterone withdrawal (Roche et al., 1999) and on the rate of progesterone clearance following CIDR removal. Short-term progesterone treatments were evaluated, because longer insertion periods have been associated with lower conception rates in C. elaphus (Fennessy et al., 1990), because treatment with progesterone for an extended period adversely affects the competency of antral follicles (McLeod et al., 2001).

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The incidence of estrus for Treatment 1 was greater than that reported for the species D. dama after treatment with CIDR® for 8 days and cloprostenol injection 48 h after the insertion of the same (Morrow et al., 1995). The difference between the incidences of estrus is probably the result of the timing of the cloprostenol administration. When administration occurs at the beginning of treatment, the developing CL (i.e. 1–5 days development at device insertion) can be refractory to the luteolytic effects of prostaglandin and luteal development continued beyond the period of the CIDR® device treatment, leading to poor synchronization rates. This observation reinforces the need for a second PGF2␣ injection at or near the end of shortterm progesterone treatments, when the CL in formation reach 8 days old and present greater capacity to respond to luteolytic stimuli (Morrow et al., 1995). The time required for the onset of behavioral estrus after the completion of Treatment 2 was similar to that previously described for the same species; signs of estrus 48–72 h after the second injection of cloprostenol with an interval of 10 days (Duarte and Garcia, 1995). However, an interval of 43–96 h for the onset of estrus signs was reported for the species D. dama (Asher and Thompson, 1989), suggesting that this interval depends on the follicular state at the time of luteolysis induction (Rubianes, 2000). One possibility of diminishing this time is to reduce the interval between PGF2␣ injections in order to affect the first-wave dominant follicle, because studies involving C. elaphus determined the sensitivity of CL to PGF2␣ from day 6 of the estrous cycle (Asher et al., 1995). Given that this period probably varies between species, studies to evaluate the period of refractoriness in CL in the species M. gouazoubira are required. The fact that premature regression of CL appears to be greater when a shorter interval between injections is used should be considered. Consideration should also be given to that fact that the time required for the onset of estrus in the two treatments could have been reduced by the presence of a vasectomized buck during monitorization. As previously described for Cervus eldi thamin in which time of estrus was synchronized with CIDR® (Hosack et al., 1999), the direct or indirect presence of bucks is capable of speeding up the preovulatory LH surge as well as the time of onset of estrus. The duration of synchronized estruses was similar to the natural estrus of the species M. gouazoubira reported by Santos et al. (2001), with a duration of 12–54.1 h, but shorter than that reported by Pereira et al. (2006) of 23–80.6 h. In contrast, the duration of the inter-luteal phases in Treatments 1 and 2 was greater than that reported for the same species in natural conditions (1.7 ± 0.1 days) (Pereira et al., 2006). As in the natural estrous cycles of brown brocket deer hinds (Pereira et al., 2006), behavioral estrus was associated with nadirs in fecal progestin concentrations and according to Patton et al. (1999), the association between nadirs in progesterone concentrations and mating behavior suggests that this measure often reflects the fertile period of an individual and not just CL regression. However, no significant increase in fecal progestins occurred from day 9 after the onset of estrus, as previously described by Pereira et al. (2006).

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The time required for the progestins to achieve interluteal concentrations, the greatest duration of this phase and number of days required for a significant increase in progesterone concentrations, suggests that PGF2␣ induced a gradual decline in fecal progestin concentrations when administered in the presence of a functional CL in Treatment 2, as previously observed in the species Oryx dammah (Shaw et al., 1995). Similarly, in Treatment 1, a slow decrease in fecal progestin concentrations occurred after the removal of the CIDR® device in association with PGF2␣ administration. The estrous behavior in hinds is associated with an increase in plasma estradiol concentration (Monfort et al., 1990). In the species M. gouazoubira (Santos et al., 2001) and in D. dama, this behavior culminates in copulation and the period of acceptance is related to a reduction in estradiol concentrations (Asher et al., 1986). However, in the present experiment, no differences in fecal estrogen concentrations were detected throughout the treatments. It is possible that the failure to detect fecal estrogen peaks could be due to the lesser concentrations of circulating estrogen or due to its excretion as a urinary metabolite, as described for other ungulate species (Schwarzenberger et al., 1996). Although the greatest values observed in the E2:P ratio throughout treatments have proved useful for detecting the time of ovulation in humans (Lenton et al., 1989) and Gazella dama mhorr (Pickard et al., 2001). In studies with other species, practically no information exists regarding the duration of estrus. Two main points could be responsible for this fact: the use of vasectomized stags to detect estrus by mating could reduce the duration of all subsequent estrous behaviors due to mechanical action of the penis against the vagina (Romano, 1993) and the evaluation of the synchronization treatments by conception rates. These show great variation (28.8–80.8%) due to numerous variables, such as: different synchronization methods, different artificial insemination routes, different semen concentrations and forms of preservation, the number of inseminations and the period in which these were performed. The lack of detections of the CL during the laparoscopy of hind B associated with luteal concentrations of fecal progestins, suggests that the progesterone could be of adrenal origin (Asher et al., 1989) or that luteal tissues were not visible (Harder and Moorhead, 1980). The repetition of behavioral estrus in hind D, with the observation of large follicles and fecal hormone profiles, suggested the occurrence of an initial ovulatory estrus involving a poor quality/hypofunctional CL that rapidly regressed, leading to a new LH surge and a second ovulation (Degliannis et al., 2005). Both these hinds presented accentuated difficulties of adaptation to captive management and could have suffered from the influence of stress, a common problem in the productive management of other deer species (Asher and Thompson, 1989; Asher et al., 1992). Greater concentrations of corticosteroids (Peters and Lamming, 1986) and progesterone produced by the adrenal gland (Asher and Thompson, 1989) can inhibit the LH surge; however, because no indication of ovarian cyst formation was observed, it is likely that the effects of stress had greater

action on follicular growth or emergence than on the induction of an inadequate LH surge (Cavalieri et al., 2002). In agreement with reports by other authors (Asher and Smith, 1987; Jabbour et al., 1993b), CL counts by laparoscopy 6 days after the end of estrus was an efficient way of evaluating the ovulation rate in this work. A greater incidence of ovulation occurred in the left ovary (66.67%), in contrast to that reported for cows (Arthur and Noakes, 1996) and sheep (Dickie et al., 1999), in which the right ovary had greater activity. Analysis of the CL produced in each treatment is a valid form of evaluating the probable superiority in fertility, because this is responsible for maintaining the initial stages of gestation (Adam et al., 1985; Pereira et al., 2006). Moreover, estrous cycle progesterone concentrations have been positively related to the volume of luteal tissue in sheep (Bartlewski et al., 1999) and O. virginianus (Harder and Moorhead, 1980). Although this relation was not observed in the present study, nonvisible portions of the CL can complicate the measurement of its true volume, provoking distortions in the volume/progesterone production relation (Harder and Moorhead, 1980). Thus, because no significant difference was observed between the mean CL diameters or between fecal progestin concentrations 6 days after the end of estrus, it was not possible to predict which of the treatments evaluated would result in the most desirable fertility. Despite studies involving D. dama reporting greater fertility indices for protocols using CIDR® compared to those using prostaglandin and its analogues (Jabbour et al., 1993a), this question requires further evaluation (Asher et al., 2000), since the reported results could be due to the poor efficiency of prostaglandin at synchronizing estrus in seasonal deer species at the beginning of the reproductive season. The greater percentage of hinds that had vulvovaginitis and purulent vaginal discharge due to the wing tips of the CIDR® device has already been described in C. elaphus and did not appear to affect fertility (McCorkell et al., 2007). 5. Conclusion In conclusion, both treatments proved capable of synchronizing estrus in the species M. gouazoubira, with the formation of functional corpora lutea, an important and decisive factor for the success of procedures like artificial insemination, embryo transfer and the application of reproduction biotechniques in the preservation of Neotropical deer species. However, the fertility of these treatments still requires direct evaluation. Acknowledgements This research was supported by CAPES, CNPq and FAPESP. The authors are grateful to Ph.D. Genner T. Pereira, for his help with the statistic analysis and M.S. Marina S. Munerato, Ana Paula C. Ribeiro for their assistance during anesthesia and surgery. The authors are also grateful to M.S. Vanessa V. Abril, M.S. Elias Alberto C. Gutierrez and the technical team of the NUPECCE, special thanks to the keepers for their assistance with the deer.

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