Author's address (for correspondence): LO Teodoro, Companhia Brasileira de Metalurgia e Mineração (CBMM), Córrego da Mata, s/n°, Caixa Postal 08, Postal Code 38.183.970, Araxá, Minas Gerais, Brazil. E-mail: firstname.lastname@example.org
The aim of the study was to evaluate the seasonality of andrological characteristics and hormonal profile of captive maned wolves (Chrysocyon brachyurus, Illiger 1811). Three adult males were evaluated from the Companhia Brasileira de Metalurgia e Mineração Scientific Breeding Center in Araxá, MG, Brazil, over 13 months. Semen was collected 2–3 times weekly and analysed. Scrotal circumference, biometrics and testicular volume were measured. Stool samples were collected 2–3 times weekly to analyse corticosteroid and testosterone metabolite concentrations. A success rate of 100% was achieved in the collection attempts during the breeding season (BS) and 77.8% during the non-breeding season (NBS). The interval to achieve penile erection was 1–5 min in the BS and 6–10 in the NBS (p < 0.001). Of the ejaculates collected, 80.0% contained sperm during BS, while 28.6% did during the NBS. The ejaculate had only one fraction, was odourless, predominantly translucent (72.4%), with a watery appearance, pH 6.7 and osmolarity of 352.8 mOsmol. Seasonal influences were seen in ejaculate volume (1.3 ml vs 0.4 ml), number of spermatozoa per ejaculate (73.9 × 106 vs 6.1 × 106) and percentage of live sperm (82.0% vs 66.1%) between the BS and NBS (p < 0.05), respectively. A high percentage of major sperm defects were observed in both seasons (50.1% in BS; 65.7% in NBS). Testicular volume was larger (p < 0.05; right testicles 13.1 cm3 in BS vs 4.0 cm3 in NBS, while left testicles 12.9 cm3 in BS vs 5.3 cm3 in NBS) and testicular consistency increased in the BS. No difference was seen in the basal faecal metabolite concentrations of testosterone; however, the corticosteroid concentrations were higher in the BS. Based on these results, it is possible to conclude that the collection of semen is feasible in captive maned wolves without compromising libido, seminal characteristics and reproductive behaviour and that sperm production is influenced by seasonality; however, it appears that there is no seasonal influence on basal testosterone concentrations.
The maned wolf (Chrysocyon brachyurus) is a solitary neotropical canid that inhabits the savannahs of South America. It is a species considered threatened, having a population that is continually declining owing to current habitat degradation and reduction, as well as high rates of roadway-related deaths (Rodden et al. 2008). The maned wolf is protected in international agreements, including Appendix II of the International Trade Convention on Wild Flora and Fauna Species Threatened by Extinction – CITES 2011.
This species has been maintained in zoos for more than 40 years (Songsasen and Rodden 2010); however, the ex situ population is considered unviable owing to low reproductive efficiency (Songsasen et al. 2006). Despite this, there are few studies on reproductive physiology that include the collection and characterization of maned wolf semen (Mascarenhas et al. 2002, 2007; Paula et al. 2002; Cunha et al. 2008; Comizzoli et al. 2009; Carneiro et al. 2011).
In the light of the scarce work on the reproductive seasonality of the male maned wolf, the aim of the present study was to evaluate the andrological characteristics, hormonal profile [faecal metabolites of corticosteroids (FMC) and testosterone] and their variation during a consecutive 13-month period in captive animals.
Materials and Methods
The research was carried out in accordance with ethical principles for research in animals and under the auspices of the Ethics Commission on the Use of Animals of the University of Franca (no024/2010, 09/09/2010).
The experiment was performed from April 2010 to June 2011 at the Scientific Breeding Center of Companhia Brasileira de Metalurgia e Mineração (CBMM) in Araxá, MG, Brazil, which is regulated by the Brazilian Institute on the Environment and Renewable Natural Resources. The breeding season was defined as the period from March to July and the NBS from August through February, in accordance with a retrospective study carried out to determine the Breeding Centers’ maned wolves’ birth periods (Borges 2005).
Three adult male maned wolves originating from the wild (n = 1) and captivity (n = 2), weighing an average of 25 kg (range 23–26 kg) and having an average age of 49 months (range 34–58 m), with no reproductive history, were studied. The animals were maintained in pairs with their respective mates in contiguous enclosures, separated by a wire mesh fence that permitted visual, olfactory and auditory contact between the pairs. Wolves 1 and 2 were part of the entire experiment while wolf 3 was admitted in February 2011.
The males were managed in their own enclosures, restrained using a yoke (Ketch All Pole, San Luis Obispo, CA, USA) and muzzle, for which all the animals had been conditioned to. Semen samples were collected by penis digital stimulation, 1–3 times weekly, from April 2010 to May 2011, with higher frequency during the breeding season. At the end of each collection, the handler compensated the animals with fruit and meat. Time intervals of 1–5, 6–10, 10–15 and 15+ min were used to measure time to erection.
Environmental temperature and humidity were measured using a Davis Wireless Vantage Pro 2 Metrological Station (Davis Instruments, Hayward, CA, USA). Data were gathered for the dates of semen collection.
Colour, consistency, odour, volume and pH of the semen were recorded. To evaluate total motility (Mot.) and determine vigour (Vig.), 5-μl aliquots of semen were placed on glass slides with cover slips and examined at ×100 magnification with light microscopy, and the results were expressed in 0–100% and scored from 0 to 5, respectively. To determine the number of spermatozoa per ejaculate (SPTZ), the semen was diluted (1 : 20) in formalin 10% buffered saline, and the cells were counted in a Neubauer chamber at ×400 magnification. The per cent of live sperm (Live) were evaluated using eosin staining (Vetec Química Fina Ltda., Rio de Janeiro, Brazil). Sperm morphology was examined on smears stained by Karras method (Papa et al. 2001), in which 200 cells were evaluated at 1000× and classified as major and minor defects (Mies Filho 1982), with results expressed in per cent.
From July 2010 to June 2011, scrotal circumference was measured monthly using a measuring tape at the median position in plane perpendicular to the long axes of the testes, and the results were expressed in millimetres. Testicular measurements of length, width and height were determined using a electronic digital caliper (model ABS scale 0–150 mm, accuracy ± 0.2 mm/.01″, setting 0.1 mm/0.01″, LOTUS, Curitiba, Brazil). Testicular volume was calculated using the formula proposed by Musson (2001), cited by Zindl (2006), and the results were expressed in cm3.
Osmolarity was measured using a steam pressure micro-osometer (VAPRO 5520; Wescor, Logan, UT, USA) on 15 samples of seminal plasma from three wolves, obtained during the breeding season and frozen at −20°C until shipped for analysis.
Fresh stool samples (80 g) were collected 2–3 times weekly during the experiment and were immediately frozen at −20°C until analysis. The assays of FMC and of testosterone (FMT) were determined by enzyme immunoassay (ELISA–Enzyme Linked Immunosorbent Assay), as described by Brown et al. (2004) and validated for FMC (Spercoski 2007) and FMT in maned wolves (R. N. Morais, Spercoski K. M., personal communication).1
Descriptive statistical analyses were performed on variables of the semen, hormonal (total and base values2), testicular biometry of all samples, by individual and by season. Samples with no volume were not included in the statistical analyses. The data that were not normally distributed (Shapiro Wilk) were analysed using the Mann–Whitney U-test, while the t-test was used in variables that had a normal distribution, to measure the difference between the groups, individuals between groups and between the individuals within each group or season, with level of significance established as 5%. Pearson's correlation was used to correlate the variables within each group. The data were processed using SigmaPlot for Windows Version 11.0 (Systat Software Inc, San Jose, CA, USA).
A success rate of 100% (70/70) was achieved in the collection of semen attempts during the breeding season (BS) and 77.8% (21/27) during NBS. Of the ejaculates collected during the BS, 80.0% (56/70) contained sperm cells, while 28.6% (6/21) did during the NBS. During collection, the wolves were calm, allowed manipulation and were conditioned to the presence of the team and of the procedure. The animals were kept in a four-footed position with the exception of wolf 2, who lay down during some procedures and was held upright by the handler who secured the chest. Even so, the animal maintained an erection in all situations. The animals’ posture was similar to domestic dogs, with an arched spine, pelvic movements and vocalization. The time interval to reach penile erection was lower (1–5 min) in the BS compared with the NBS (6–10 min; p < 0.001). It was obtained from the first collection on all attempts both within and out of the BS with the exception of one sample of wolf 2 in the NBS.
The collected ejaculates had only one fraction, the colour of which varied from translucent (72.4%), whitish (10.8%) to white (8.6%). Some samples were yellow (6.5%) or red (1.1%) in colour. Seasonal variation in the colour of the samples was seen, with a larger number of samples being translucent and whitish in the BS and translucent in the NBS (p = 0.039). The samples had a watery appearance. The semen was odourless, except when contaminated by urine. Some samples of ejaculate contained calcium crystals, which were visible on the fresh semen slides.
The results of the seminal characteristics are presented in Table 1, including samples with concentration equal to zero. The SPTZ varied from 0 to 378.8 × 106 in the BS and from 0 to 13.5 × 106 in the NBS. Table 2 details the results of sperm morphology in the 55 ejaculates that had sperm cells.
Table 1. Mean (standard deviation) of the seminal characteristics during the breeding season (BS) and non-breeding season (NBS), collected from April 2010 to April 2011 from three maned wolves
All (n = 70)
Wolf 1 (n = 25)
Wolf 2 (n = 31)
Wolf 3 (n = 14)
All (n = 21)
Wolf 1 (n = 10)
Wolf 2 (n = 10)
Wolf 3 (n = 1)
Different letters in same row indicate significant differences between wolves within the same season (p < 0.05).
Statistically significant difference between evaluated variable of all samples as a whole and in the same animal by season (p < 0.05).
The results of testicular volume and scrotal circumference are shown in Table 3. Although the evaluation of testicular consistency was not part of the present study, firmness was seen in the BS, while flaccidity was observed in the NBS.
Table 3. Mean (standard deviation) of the volume of right testicle (RTV) and left (LTV) and scrotal circumference (SC) from three maned wolves in breeding season (BS) and non-breeding season (NBS)
All (n = 14)
Wolf 1 (n = 5)
Wolf 2 (n = 5)
Wolf 3 (n = 4)
All (n = 15)
Wolf 1 (n = 7)
Wolf 2 (n = 7)
Wolf 3 (n = 1)
Different letters in same row indicate significant differences between wolves within same season (p < 0.05).
Statistically significant difference between evaluated variable of all samples as a whole and in the same animal by season (p < 0.05).
The average value and the standard deviation of seminal plasma osmolarity of all the samples was 352.8 mOsmol ± 17.4 (n = 15), and individual results were 337.3 mOsmol ± 6.8 (n = 3) in wolf 1; 362.8 mOsmol ± 13.7 (n = 5) in wolf 2 and 351.5 mOsmol ± 22.1 (n = 6) in wolf 3. Total and basal concentrations of faecal corticosteroid and testosterone metabolites are presented in Table 4.
Table 4. Mean (standard deviation) of the concentration of total and basal faecal metabolites of corticosteroids (FMC) and of testosterone (FMT) during the breeding season (BS) and non-breeding season (NBS) in three maned wolves
All (n = 86)
Wolf 1 (n = 35)
Wolf 2 (n = 35)
Wolf 3 (n = 16)
All (n = 60)
Wolf 1 (n = 30)
Wolf 2 (n = 30)
Different letters in same row indicate statistical differences between wolves in same season.
Difference between evaluated variables in same animal in and out of breeding season (p < 0.05).
A significant difference (p < 0.01) was seen in the environmental temperatures between seasons (20.5°C ± 1.9 in BS vs 21.8°C ± 2.1 in NBS), although there was no difference in the humidity (75.7 mm ± 13.1 in BS vs 63.4 mm ± 18.0 in NBS). These data were not correlated with any variables of semen quality.
Several variables in the general and seminal morphological analyses were correlated with faecal metabolite concentration. In the BS, there was a negative correlation between the volume of the ejaculate and the concentration of faecal testosterone metabolites (r = −0.37; p = 0.006) and between time to achieve erection and concentration of faecal testosterone metabolites (r = −0.40; p = 0.003). During the NBS, there was a positive correlation between the concentration of faecal testosterone metabolites and total spermatozoa concentration in the ejaculate (r = 0.45; p = 0.007).
Semen collection was easy to perform, obtaining material from the first attempt. This method of semen collection is not commonly described for wild canids owing to behavioural and safety issues (Shaw 1975; Paula et al. 2002; Minter and Deliberto 2005) and the need for prior male conditioning to perform the collection (lasting up to 1 year). Furthermore, wild canids can alter their behaviour in the presence of humans (Mascarenhas et al. 2002; Carneiro et al. 2011). The procedure adopted in the present study maintained the behavioural characteristics of the species, in that they mated with females throughout the BS of the study period. The only aspect of the study conditions that was different was related to the physical restraints with yoke and muzzle, but to which the wolves from the Breeding Center had previously been exposed to for other procedures.
Maned wolf ejaculate differs from domestic dogs in that we observed only one fraction of the ejaculate contrary to the findings of Mascarenhas et al. (2007). The semen pH was similar to that reported in the domestic dog (England and Allen 1989), and this was not affected by seasonality, although there was individual variation seen between wolves in the BS. Osmolarity of maned wolf semen has not been described in the literature; values varied from 321 to 388 mOsmol, with an average of 352.8 mOsmol, similar to the osmolarity of red wolf semen (Canis rufus; Lockyear et al. 2009) and higher than that of the dog (~310 mOsmol; Hay et al. 1997). These results indicate the need to develop an appropriate extender to preserve maned wolf semen, bearing in mind that adjusting the osmolarity to the semen extender is critical to avoid osmotic stress (Martínez-Pastor et al. 2006).
Seasonal influence was seen in the volume of ejaculate, number of spermatozoa per ejaculate and percentage of live cells, which were all higher, with overall superior quality of the ejaculate in the BS, compared with the NBS.
Coinciding with increased testicular firmness, testicular volume was significantly larger in the BS, indicating seasonality in this variable. The drastic reduction in sperm production shortly after the BS explains the reduced testicular volume. It was confirmed that even in the males that had testicular volume reduction immediately after the reproductive season, the capacity to return to testicular development in the subsequent BS was maintained, just as Forsberg et al. (1989) observed in foxes (Vulpes vulpes).
No seasonal difference was observed in the basal FMT concentrations, between BS and NBS, but there was a difference in total values, with higher concentrations in the BS. This finding should be viewed cautiously because one animal (wolf 3) entered the study at the end of the experimental phase and therefore only contributed stool samples during the BS. When wolf 3 data were removed from the total values (data not presented), there was also no difference found regarding total FMT concentrations. Stable concentrations of testosterone across seasons seen in Lycaon pictus (Johnston et al. 2007) and other wild canids (Asa 1999) suggest that sperm production and testicular volume are independent of the secretion of testicular testosterone.
In the present study, there was a high percentage of abnormal sperm both during the BS and NBS (Fig. 1) similar to that observed by Carneiro et al. (2011) and Mascarenhas et al. (2007). It is feasible that this is the result of inbreeding; however, the inbreeding coefficients of the two captive-born wolves were low at 0.00 and 0.13 for wolves 2 and 3, respectively (R. Holland 2011, verbal communication)3 and cannot be considered a factor that had an impact on the results. Despite the high per cent of defects found in the present study, wolf 3 bred with its mate during the 2011 BS, and this resulted in a confirmed pregnancy. This high percentage of defects may be a characteristic of the maned wolf, as has been observed in other wild species (Morato et al. 2001; Minter and Deliberto 2005).
Seasonal differences were seen in the FMC analyses, as found by Spercoski (2007), who reported seasonality in adrenocortical function in free-living wolves. When data were examined from individuals, the FMC in wolf 1 was higher during the BS and was associated with a low concentration of FMT, but the same pattern was not seen in the other two animals. This higher concentration of FMC could explain the poor semen quality of wolf 1, independent of season, with low number of spermatozoa per ejaculate and high percentage of sperm with major defects, despite a higher volume of semen (Rivier and Rivest 1991; Genuth 2004).
The current work is the first to describe the influence of seasonality on maned wolf sperm production. The confirmation that semen collection can be achieved in this species opens prospects for more profound studies on seminal characteristics, as well as the development of protocols for freezing semen from captive animals and the establishment of germplasm banks, for ex situ species conservation.
This study was performed within the framework of the master program of University of Franca, Brazil, and of the Companhia Brasileira de Metalurgia e Mineração – CBMM, Araxá, Brazil. All financial support was provided by CBMM. The analysis of maned wolf semen osmolarity was performed by professor Carolina Arruda Freire of the Laboratory of Comparative Physiology of Osmoregulation, Biological Sciences Sector, Polytechnical Center, Federal University of Paraná.
Conflicts of interest
None of the authors have any conflicts of interest to declare.
This manuscript is the result of Laura Teodoro de Oliveira's Master thesis, under the guidance of Fabiana Ferreira de Souza. Both Laura and Fabiana conceived the study, analysed the data and prepared the manuscript. Laura performed all the semen collections and laboratory sample analyses. Katherine Maria Spercoski carried out the corticosteroid and testosterone faecal metabolites evaluations. Rosana Nogueira de Morais oversaw the experimental phase of the collection and analysis of the faecal steroid hormonal metabolites.
Information obtained on 06-May-2011 from Professor Dr. Rosana Nogueira de Morais, adjunct professor, Department of Physiology, Federal University of Paraná and Katherine Maria Spercoski, MS, doctoral student, Department of Physiology, Federal University of Paraná.
Average values were calculated (±standard deviation, SD of the mean) for FMC and FMT levels for each analysed group, after the exclusion of values that exceeded the average ±1.5 SD, which were considered spikes (Songsasen et al. 2006).
Information obtained on 23 Aug 2011 from Ruben Holland – Curator of the International Maned Wolf (C. brachyurus) Studbook, Zoo Leipzig GmbH, Pfaffendorfer StraBe 29, D-04105 Leipzig.