Reproductive aspects of male franciscana dolphins (Pontoporia blainvillei) off Argentina

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright

Author's personal copy Animal Reproduction Science 131 (2012) 41–48

Contents lists available at SciVerse ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Reproductive aspects of male franciscana dolphins (Pontoporia blainvillei) off Argentina María Victoria Panebianco a,∗ , María Fernanda Negri a,1 , Humberto Luis Cappozzo a,b a Laboratorio de Ecología, comportamiento y mamíferos marinos (LECyMM), Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” MACN-CONICET; Av. A. Gallardo 470, C1405DJR Buenos Aires, Argentina b Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y Diagnóstico (CEBBAD), Fundación de Historia Natural Félix de Azara Departamento de Ciencias Naturales y Antropología, Universidad Maimónides, Hidalgo 775 piso 7, C1405BDB, Buenos Aires, Argentina

a r t i c l e

i n f o

Article history: Received 25 November 2011 Received in revised form 31 January 2012 Accepted 2 February 2012 Available online 11 February 2012 Keywords: Pontoporia Reproductive biology Sexual maturity Testis South America Argentina

a b s t r a c t As the first study to investigate reproductive aspects of male franciscana dolphin – Pontoporia blainvillei – in Argentine waters, the aim of this paper was to assess sexual maturity by using histological and morphometric methods. P. blainvillei was labeled as “Vulnerable” by the IUCN in 2008. The specimens analyzed were either incidentally caught in artisanal fishing nets (n = 47) or found stranded on the beach (n = 3). Testis weight and testicular index of maturity were reliable indicators of sexual maturity, being their values: MTW: 1.14 ± 0.60–4.49 ± 1.94; IM: 0.03 ± 0.01–0.09 ± 0.03, for immature and mature specimens’ respectively. It was found that the hierarchical cluster analysis (HCA) might be appropriate for establishing sexual maturity stage, based on both the body morphometric measurements and age. The values for age, standard length and total weight at attainment sexual maturity were 2.92–3.54 years, 126.19–126.27 cm and 23.47–23.75 kg. Considering the extremely low relative testis weight, the reversed sexual length dimorphism, the absence of secondary sexual characteristics, and the lack of scars from intrasexual combats in males, the hypothesis that sperm competition does not occur in the species, and male combats for accessing female reproductive may be rare for P. blainvillei is reinforced. All these features fit the species within a serial monogamous mating system. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Franciscana dolphin, Pontoporia blainvillei, inhabits coastal waters of the western South Atlantic Ocean, from Itaúnas (18◦ 25 S-30◦ 42 W), Brazil (Siciliano, 1994), to Golfo Nuevo (42◦ 35 S-64◦ 48 W) Argentina (Crespo et al., 1998). P. blainvillei accidentally dies captured in artisanal coastal fishing nets. An estimation of 500–600 individuals for

∗ Corresponding author. Tel.: +54 11 4982 9410x6595; fax: +54 11 49825243x4494. E-mail addresses: [email protected], [email protected] (M.V. Panebianco), [email protected] (M.F. Negri), [email protected] (H.L. Cappozzo). 1 Tel.: +54 11 4982 9410x6595. 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2012.02.005

the Southwestern Atlantic coast of Buenos Aires has been recorded for that cause, representing 2.5–3.6% of the population of Argentina (Bordino and Albareda, 2004; Cappozzo et al., 2007; Corcuera et al., 1994; Crespo et al., 1998; Negri, 2010). International Whaling Commission establishes an upper limit of 2% of mortality for the species to be sustainable. The International Union for Conservation of Nature considers P. blainvillei as “Vulnerable” throughout its distribution range, due to the high risk of extinction it faces in the wild (IUCN, 2010). Some reproductive aspects of this dolphin, as estimation of age at sexual maturity, description of the reproductive cycle, and histological descriptions of its gonads, were previously described for the coastal zone of Brazil and Uruguay (Harrison et al., 1981; Harrison and Brownnell, 1971; Harrison et al., 1981; Kasuya and Brownell, 1979;

Author's personal copy 42

M.V. Panebianco et al. / Animal Reproduction Science 131 (2012) 41–48

Brownell, 1989; Danilewicz, 2003; Danilewicz et al., 2004; Botta, 2005; Bertozzi, 2009). In Argentina, Corcuera and Monzón (19901 ), Monzón and Corcuera (19902 ) and Corcuera (19963 ) presented the only available results on reproductive aspects for P. blainvillei of the southernmost of the distribution range. Whether the incidental mortality suffered by P. blainvillei is a threat to the species population will depend on its relative magnitude of the potential rate of increase. Understanding the population dynamics of species is an important step to develop conservation strategies and comparative biology studies. Particularly, estimation of age and length at sexual maturity might provide significant data about fluctuations and trends of the population (Botta, 2005; Caswell et al., 1998). 2. Materials and Methods 2.1. Sample data Specimens (n = 47) collected during the reproductive season (November–March) between 1998 and 2010 were analyzed, they were either incidentally caught in artisanal fishing nets or found stranded on the beach (n = 3). The study area embraces the coastal zone of the Southern Buenos Aires, between the localities of Necochea (N, 38◦ 37 S, 58◦ 50 W) and Bahía Blanca (BB, 38◦ 44 S, 62◦ 14 W) (Fig. 1). The carcasses of franciscana dolphins were brought to port by fishermen or found on the beaches and frozen prior to necropsy. Recorded information included sex (determined externally), total weight (TW, n = 50), standard length (SL, n = 48), and the presence of scars and wounds. 2.2. Age determination In order to ascertain the age, the protocol of Pinedo and Hohn (2000) was followed with modifications ad hoc. Teeth were extracted from each specimen (n = 48) and then fixed in a 10% formaldehyde solution. Subsequently, they were decalcified in commercial acid mix RDO® and sectioned in a cryostat at −21 ◦ C. Sections of 25 ␮m were stained with Mayer’s hematoxylin and mounted in slides with glycerine. Age was determined by counting growth-layer groups (GLGs). Only central well-contrasted layers were used for GLG readings in both dentine and cement. Although no direct validation exists for GLGs, indirect evidence supports that one GLG represents one year of age (Kasuya and Brownell, 1979; Pinedo, 1991; Pinedo and Hohn, 2000).

1 Corcuera, J., Monzón, F., 1990. Reproducción en hembras del delfín franciscana (Pontoporia blainvillei) en Necochea (Argentina). Abstracts of the IV Reunión de Trabajo de Especialistas en Mamíferos Acuáticos de América del Sur, Valdívia, Chile, p. 20. 2 Monzón, F., Corcuera, J., 1990. Análisis preliminar de madurez sexual en machos de delfín franciscana (Necochea, Provincia de Buenos Aires, Argentina). In 7th Workshop of Specialists in Aquatic Mammals of South America, Valdivia, Chile, p 46. 3 Corcuera, J., 1996. Edad de madurez sexual del delfín Franciscana en el Sur de la Provincia de Buenos Aires Argentina. Abstracts of the VII Reunión de Trabajo de Especialistas en Mamíferos Acuáticos de América del Sur, ˜ del Mar, Chile, p. 39. Vina

Fig. 1. Sampling localities along the Southern coast of Buenos Aires Province.

2.3. Testis histology To perform a histological analysis only the unspoiled testis sample could be inspected (n = 28). The diminishing in the samples was caused either by animals eating the gonads of stranded animals or the high decomposition of the tissue. For each gonad a subsamples of 3 mm thickness were obtained from the middle of each testicular piece. Each slice was dehydrated gradually in alcohol baths of increasing concentrations (80%, 96% and 100%) and then immersed in pure xylene. After dehydration the tissue was embedded in Histoplast® (Biopack) and the blocks obtained were cut with a digital microtome into 4–7 ␮m thick sections which were stained with hematoxylin–eosin. Male sexual maturity stage was determined through observation of 10 seminiferous tubules randomly selected for each testis sections. According classification criteria for sexual maturity stage suggested by Hohn et al. (1985) and Danilewicz et al. (2004), the specimen can be listed on: Immature: seminiferous tubules containing mainly spermatogonias and spermatocytes. Abundant interstitial tissue present between the seminiferous tubules and lumen totally closed. Mature: seminiferous tubules containing spermatogonias, spermatocytes, and, in many cases, spermatids. Interstitial tissue almost non-existent between the seminiferous tubules. The lumen is, often, totally opened. 2.4. Body morphology Estimation of sexual maturity was obtained by analyzing jointly the parameters most commonly used for such

Author's personal copy M.V. Panebianco et al. / Animal Reproduction Science 131 (2012) 41–48

determination (SL, TW and age). SL was measured as a straight line from the tip of the rostrum to the fluke notch (American Society of Mammalogists, 1961), and TW was obtained by an analog dynamo (±0.002 gr). Since not all the information could be collected from each dolphin, the sample sizes for each parameter diverge according to the carcass condition. Two hierarchical cluster analysis (HCA) based on the morphometrics parameters SL, TW and age were conducted, the first one for those dolphins which gonads were no collected (n = 17) labeled Dataset A, and a second one, with the objective of validate the results previously obtained, for those whose maturity stage was assessed by histological method (n = 24) labeled Dataset B. Mean values of morphometrics parameters corresponding to the each maturity group (Immature and Mature) were statistically compared between those from Dataset A with those from Dataset B (e.g. mean values of SL for Mature from Dataset A were compared with mean values of SL for Mature from Dataset B). In order to define the best linkage method for the HCA, the highest value of the cophenetic correlation coefficient was applied. Thus, the furthest neighbor linkage method with Euclidean distance was selected for both HCAs. To perform the HCAs the statistic software InfoStat/E (Di Rienzo et al., 2010) was used. The goal was to create a hierarchy of methods to use for inferring state of sexual maturity when histological data were not available.

2.5. Gonad morphology Testis (n = 38) were separated from the epididymis, weighted (±0.01 gr) and measured (±0.01 mm) in three dimensions (length, width and thickness) with digital caliper. Afterward, gonads morphometric parameters were assessed for each specimen by following the criteria developed by Danilewicz et al. (2004) and Botta (2005): combined and mean testicular length (CTL and MTL), combined and mean testicular weight (CTW and MTW), mean testicular diameter (MTD), testicular index of maturity, and percentage of relative testicular weight (%RTW) – only for by-caught dolphins – (Table 1). Sexual maturity was estimated by histological methods (n = 26) and hierarchical cluster analysis (n = 18). Mean values of gonads morphometrics parameters corresponding to the each maturity group (Immature and Mature) were statistically compared by the corresponding statistical analysis. The percentage of overlapping between maturity groups (Immature and Mature) for each gonad morphometric parameter was calculated according to the following equation:

% overlaping =

Minx Mi − MaxIi Maxx Mi − Minx Ii

where Min/Max: minimum or maximum value of the x gonadal parameter; I/M: Maturity stage (Immature or Mature) of the i specimen

43

2.6. Estimation of mean age, length and weight at attainment sexual maturity To determine age, standard length and total weight – for by-caught dolphins – at which specimens attain sexual maturity, two different methods were used: logistic regression and DeMaster (1978) modified by Ferrero and Walker (1999). The logistic method was performed applying 100 iterations, by means of the non-linear Quasi–Newton method, minimizing the residual sum of squares (Cox and Snell, 1989 cited in Ferrero and Walker, 1999). The relationship between the parameters was expressed according to the equation: Y=

1 (1 + e(−xb+a) )

where x is the age, length or weight of the animal, Y is the proportion of mature and immature animals, b is the regression coefficient; and a is the y-intercept. According to DeMaster (1978) with modifications, age at sexual maturity (ASM) was expressed as the following equation: ASM =

k a=j

a(fa − fa−1 )

where fa is the fraction on animals sexually mature of the age class a, j is the age of the youngest sexually mature animal from the sample; and k is the eldest sexually immature animal from the sample. The variance of ASM was expressed by the next equation: VAR(ASM) =

k  a(1 − fa )  a=j

Na − 1

where Na is the total number of animals of age a. Length (LSM) and weight (WSM) at sexual maturity were also calculated using the equation of DeMaster (1978) modified by Ferrero and Walker (1999). The equation used for the calculation was the following:

Cmax LSM = C(ft − ft−1 ) WSM Cmin where Cmax represents the length/weight class of the sexually immature specimens with the highest length or weight, Cmin is the length/weight class of the sexually mature specimens with the lowest length or weight, C is the smallest value of the length/weight class t, and ft is the fraction of sexually mature animals from the length/weight class t. Specimens were grouped into intervals of 5 cm and 5 kg, to minimize the error in the analysis. The calculation of the variance was expressed by the equation: VAR(LSM/WSM) = w2

k  



f (1 − ft )/Nt − 1

a=j

where Nt is the total number of animals of the length/weight class t, and w2 is the interval of classes, which in this case was 5. All statistical analysis applied in this study (Shapiro– Wilk’s test for normality, Levene’s homocedasticity

Author's personal copy 44

M.V. Panebianco et al. / Animal Reproduction Science 131 (2012) 41–48

Table 1 Gonads morphometric parameters calculated for male franciscana dolphins, Pontoporia blainvillei off southern Buenos Aires coast. Parameters

Acronym

Description

Units

Combined testicular length Combined testicular weight Mean testicular length Mean testicular weight Mean testicular diameter Testicular index of maturity Relative testicular weight

CTL CTW MTL MTW MTD IM RTP

Sum of the length of the left and right testis of each dolphin Sum of the weights of the left and right testis of each dolphin Mean length of the testis of each dolphin Mean weight of the testes of each dolphin Mean diameter of the testes of each dolphin Relationship between the CTW and the sum of lengths of the left and right testis Relationship between the CTW and the TW

cm gr cm gr cm gr/cm %

test, comparison tests: Kruskal–Whallis and Mann–Whitney, Wilcoxon and Spearman’s ranks test) were performed with the statistical software Statistica 7.0 (Statsoft, Inc.) and InfoStat. For all tests performed the level of statistical significance was set at P ≤ 0.05 and the data presented as mean ± SD. 3. Results 3.1. Testis histology Sexually immature males (n = 16) showed the transversal section of the seminiferous tubules with a circular shape, these seminiferous tubules were densely embedded within interstitial tissue (Fig. 2A and B). Within seminiferous tubules which lumen was either small or completely closed, spermatogonia prevailed on their inner walls. In contrast, mature males (n = 9) showed more stretched tubules, and sparsely embedded on the interstitial tissue; within the seminiferous tubules spermatids cells were found with different maturation stages (Fig. 2C–F). The largest immature male flaunted a length of 135.2 cm, a weight of 23 kg and was three years old, while the smallest mature male displayed a length of 119.3 cm, 16.53 kg and was five years old. This last specimen showed a smaller standard length than the average for mature specimen, according the results in the present study (Table 1). 3.2. Body morphology According to HCA based on Dataset B, the sexually immature group was comprised of neonates and pubertal animals, all of which were immature according to histological standard. Differences were identified in the determination of sexual maturity between the two analyses–histological and HCA–although these differences were no significant (range: SL 119.0–122.9 cm; TW 18.25–23.00 kg; age 2–4 years; n = 5). The origin of those differences was identified as a result of an erroneous classification of five dolphins in the sexually mature group in the HCA, when in fact they belonged to the sexually immature group according to the histological analysis of their gonads. According to HCA based on Dataset A, there were two main groups containing sexually immature and mature specimens (linkage distance of 40%, and a cophenetic correlation value of 0.80; Fig. 3). The classification of immature and mature group was made by contrasting the range values of SL, TW and age, obtained from previous histological analysis (Table 2). Afterward, the mean values

of the morphometrics parameters obtained in both analysis for the two maturity groups (I/M) were compared (e.g. the sexually immature group obtained in HCA, with the same obtained in histology analysis); no differences were obtained between both analysis (Mann–Whitney: Immature: SL P = 0.64, TW P = 0.43, age P = 0.88; Mature: SL P = 0.09; TW P = 0.14; age P = 1.00). According to the previous analysis, the eldest immature specimen was four years old, 120.04 cm and 24.90 kg, whereas the youngest mature was two years old, 128.91 cm and weighted 22.07 kg (Table 1). 3.3. Gonads morphology A distinctive characteristic of Franciscana’s testicles is their small length, even for mature specimens (Table 3). Right and left gonads were symmetric, i.e. all measurements made on the testes evidenced similar values: weight (right: 2.22 ± 2.16 cm, left: 2.32 ± 2.90 cm) length (right 3.65 ± 0.97 cm, left: 3.64 ± 1.04 cm, n = 37), width (right: 0.91 ± 0.49 cm, left: 0.99 ± 0.62 cm, n = 38) and thickness (right: 0.63 ± 0.32 cm left: 0.63 ± 0.31 cm, n = 35) (Wilcoxon: weight: P = 0.69; length: P = 0.82; width: P = 0.62; thickness: P = 0.91). No significant differences were found between the gonads parameters in both methods – HCA and histological – according to the sexual maturity stage. Furthermore, all gonads variables analyzed were different between immature and mature individuals (Mann–Whitney: MTW P = 0.001; IM P = 0.001; CTW P = 0.003; MTD P = 0.005; MTL P = 0.007; CTL P = 0.010; %RTW P = 0.020). Subsequently, the relationships between standard length and the gonads parameters measured were analyzed (the parameters were pooled together since there were not significant differences between the two methods used). The values of the Spearman’s correlation coefficient, in descending order, were: IM (rs = 0.77, P ≤ 0.001, n = 35), MTW (rs = 0.77, P ≤ 0.001, n = 37), MTL (rs = 0.73, P ≤ 0.001, n = 37), CTW (rs = 0.72, P ≤ 0.001, n = 37), and CTL (rs = 0.63, P ≤ 0.001, n = 38). For MTD (rs = 0.48, P = 0.003, n = 35) a positive relationship with standard length was observed, its r value was the smallest one observed nonetheless, followed by RTW (rs = 0.56, P = 0.001, n = 37). IM and RTW showed a slightly overlapping between the two maturity groups analyzed (0–1%), while the MTW presented a 2% overlap. All the rest of the parameters showed higher percentage value (≥20%). Moreover, a qualitative increment on the values for all the parameters were observed for the specimens aged four years plus, or 125 cm (approximately); nevertheless, this was not statistically obtained.

Author's personal copy M.V. Panebianco et al. / Animal Reproduction Science 131 (2012) 41–48

45

Fig. 2. General aspect of immature (A–B) and mature testes (C–F) of Pontoporia blainvillei. Figures E and F shown spermatids (EP) at different stages of maturation present in mature testis (A: 1000×). It can be seen the conspicuos interstitial tissue and the smaller length of seminiferous tubules (A–B) of immature compared to mature individual (C–D). Scale 500 and 50 ␮m.

3.4. Estimation of mean age, length and weight at attainment sexual maturity The range of standard lengths less than 109 cm and 1year-old of age included only immature specimens, while from the range greater than 130 cm and 5 years old only mature specimens were observed. The outcomes for age, length and weight at attainment sexual maturity were: For logistic adjustment method: 2.92 ± 0.30 years (R = 0.76, explained variance 57.6%, P ≤ 0.001, n = 40), 126.27 ± 3.97 cm (R = 0.64, explained variance 41%,

P ≤ 0.001, n = 40) and 23.47 ± 1.32 kg (R = 0.52, explained variance 27%, P ≤ 0.001, n = 40). For DeMaster (1978) method: 3.54 ± 0.17 (IC 95% = 2.72–4.36 years, n = 42), 126.19 ± 0.18 (CI 95% = 125.36– 127.01 cm, n = 38) and 23.75 ± 0.18 kg (CI 95% = 22.92–24.48 kg, n = 39). 4. Discussion The distribution range of franciscana dolphins’ population under study spreads out through a highly heterogeneous climatic range, from the cold waters of

Author's personal copy 46

M.V. Panebianco et al. / Animal Reproduction Science 131 (2012) 41–48

Fig. 3. Hierarchical cluster analysis showing the maturity groups formed using morphometric Dataset A, specimens with no histological gonads data available. Dolphins codes in bold correspond to the sexually mature specimens.

central Argentina to warmer waters of Southeastern Brazil. Having invested vast effort in order to investigate how this unusual species challenge the effects of growing human population, there is still a need for extra information on biological parameters that describe the life history of the southernmost population (e.g. ASM, sexual maturity, reproductive rate). The determination (or a better understanding) of the parameters of the different populations’ species involved on this response (population growth, density-dependence) might help to discern the evolutionary processes that might be occurring. Lateral symmetry in testes’ mass of franciscana dolphins in Southern Buenos Aires shown the same result as those previously reported of dolphins from Brazil and Uruguay (Kasuya and Brownell, 1979; Danilewicz et al., 2004; Botta, 2005; Bertozzi, 2009). The histological observations on testis sections were in accordance with those

previously made by Botta (2005), who analyzed 80 gonads and was not able to visualize spermatozoa. Nevertheless, in carcasses, observation of spermatogenetic cells is a reliable way to determine maturity, but in poor condition testes they may be difficult to recognize or find. Regarding the gonads morphometrics measurement, suitable results for determination of sexual maturity were obtained by, even when spermatogenesis could not be recognized or when the testes presented poor tissue condition. In congruence with the outcomes obtained from the analysis of those morphometric parameters, it is possible to sustain that the testicular index (IM), relative testicular weight (RTP) and the median testicular weight (MTW) were reliable for establish the sexual maturity of P. blainvillei. This result was supported by the absence or low percentage of overlapping found between maturity groups for the three parameters. Similar result for maturity testicular

Table 2 Body morphometric parameters of franciscana dolphins (Pontoporia blainvillei) analyzed obtained by histological and hierarchical cluster analysis methods. There were no significant differences for the morfometrics parameters between the maturity groups analyzed according to the methods applied. Histological method

Hierarchical cluster analysis

Immature

Mature

Immature

Mature

Length (cm)

Mean SD Range 95% CI n

109.49 15.19 81.50–135.20 101.39–117.58 16

128.96 7.11 119.30–139.00 122.72–132.60 12

114.02 10.12 108.36–120.04 110.75–118.33 14

133.43 4.35 127.50–137.49 129.79–137.07 8

Weight (kg)a

Mean SD Range 95% CI n

18,5 4.53 9.87–23.00 14.82–20.59 13

22.93 2.57 16.53–26.00 19.85–24.82 8

19.52 3.92 12.00–24.40 17.24–21.76 14

25.70 4.27 22.07–35.20 22.13–29.27 8

Age (year)

Mean SD Range 95% CI n

1.60 1.24 0.00–4.00 0.91–2.29 15

4.44 1.74 2.00–7.00 3.22–5.89 10

1.64 1.01 0.00–4.00 1.02–2.18 14

5.43 3.87 2.00–13.00 2.05–8.21 8

SD: standard deviation; CI: confidence interval. a Weight data belong to by-caught dolphins.

Author's personal copy M.V. Panebianco et al. / Animal Reproduction Science 131 (2012) 41–48

47

Table 3 Gonads morphometric parameters evaluated according to the sexual maturity of Pontoporia blainvillei. There were no significant differences for the gonads parameters between the maturity groups analyzed according to the method applied. Maturity State

Parameters

Histological method

Hierarchical cluster analysis

Mean

SD

I

MTD (cm) CTW (gr) CTL (cm) IM (gr/cm) % RTW MTW (gr) MTL (cm)

0.63 2.63 6.38 0.03 0.01 1.14 3.18

0.36 2.00 1.26 0.01 0.00 0.60 0.60

0.06–1.58 0.40–8.70 4.48–9.00 0.01–0.06 0.00–0.02 0.02–2.50 2.24–4.50

M

MTD (cm) CTW (gr) CTL (cm) IM (gr/cm) % RTW MTW (gr) MTL (cm)

1.15 7.76 8.89 0.09 0.04 4.49 4.47

0.35 1.79 0.99 0.03 0.01 1.94 0.65

0.88–1.95 5.50–10.80 6.85–9.84 0.06–0.16 0.03–0.07 2.75–8.55 3.43–5.37

Range

Mean

SD

16 16 16 16 16 16 17

n

0.58 1.78 6.06 0.03 0.01 0.89 3.03

0.25 0.78 0.67 0.01 0.00 0.39 0.34

Range 0.08–0.77 0.40–2.80 5.07–6.71 0.01–0.04 0.00–0.01 0.20–1.40 2.54–3.36

n 6 8 8 8 8 8 8

8 8 8 7 7 7 8

0.96 7.60 8.21 0.09 0.03 4.53 4.50

0.42 5.80 2.42 0.05 0.02 4.27 1.35

0.37–1.66 2.60–17.10 4.50–10.73 0.06–0.19 0.01–0.06 1.30–13.00 3.10–7.01

5 5 5 5 5 5 5

I: sexually Immature individual; M: sexually Mature individual, SD: standard deviation; n: sample number; MTD: mean testicular diameter; CTW: combined testicular weight; CTL: combined testicular length; IM: testicular index of maturity; % RTW: percentage of relative testicular weight; MTW: mean testicular weight; MTL: mean testicular length.

index was previously found in population of Franciscanas from Rio Grande do Sul and San Pablo (Student’s test: RGS: t = 1.47, d.f. = 43, P = 0.15; SP: t = 0.00, d.f. = 33, P = 1.00; Botta, 2005; Bertozzi, 2009). Concerning the others gonads morphometrics parameters analyzed, such as mean and combined testicular length (MTL and CTL), a high percentage of overlap between maturity groups were found (>30%), hence, these parameters were unreliable for the estimation of sexual maturity stage. Similar results were obtained by Botta (2005) for franciscana dolphin off Brazil. For the same cause of overlapping, mean testicular diameter (MTD) was no accurate for Argentineans dolphins, although the same parameter was proved to be reliable for the determination of maturity stage for the franciscanas studied by Botta (2005). Since obtaining fresh gonads tissue for histological analysis is usually inaccessible, it is essential to provide other variables or parameters that were not affected by the carcasses’ condition from which the sexual maturity can be estimated. External morphometric measurements, such as standard length and total weight, are usually easier to measure when the specimen is survey long after death. In this regard, the versatility of hierarchical cluster analysis based on these body morphometric measurements was verified by comparing the results obtained in both HCA and histological analysis of gonads. This analysis enabled a clear distinction between sexually mature and immature specimens using body morphometric parameters as standard length, total weight and age. Yet, a range of standard length, total weight and age (range: SL 119.0–122.9 cm; TW 18.25–23.00 kg; age 2–4 years; n = 5) in the HCA returned unreliable results. For that reason, it is recommended for individuals within those ranges a ratification of sexual maturity stage by histological or morphometrical analysis of their gonads. In relation with length and age at attainment sexual maturity, Ramos et al. (2000) found a LSM of 115 cm and ASM of two years for Rio de Janeiro dolphins, while for the state of São Paulo, Brazil, Bertozzi (2009) reported a

length at sexual maturity between 103.6 and 107.8 cm and an age of 1.3 and 1.5 years. Continuing southward, for the State of Rio Grande do Sul, Brazil, an ASM of 3.6 years and a length of 128.2 cm was reported (Danilewicz et al., 2004). Male dolphins from the Uruguayan coasts showed an age at attainment sexual maturity between 2 and 3 years, and a length of 131.4 cm (Kasuya and Brownell, 1979). Previous studies off Argentina (Monzón and Corcuera, 19904 ; Corcuera, 19965 ) reported an ASM of 3.3 years and a LSM of between 125 and 135 cm, and, finally, the current study found a LSM of 126.19–126.26 cm and a maturity age of 2.92–3.54 years old. Values reported here were similar to those obtained for the dolphins of the coasts of Uruguay and Rio Grande do Sul and higher than those off Rio de Janeiro and São Paulo. From the comparison of mean standard lengths and ages at attainment sexual maturity for the different population that exist throughout the species’ distribution range, it was possible to corroborate what was described by Pinedo (1991) and genetically ratified by Secchi et al. (1998), the existence of at least two different ecotypes of the species. The relatively small size of the testes of sexually mature dolphins (4%), the absence of external scars caused by congeners, and the absence of male secondary sexual characters reinforce the outcomes exposed by Danilewicz et al. (2004) and Rosas and Monteiro-Filho (2001) for franciscana dolphins in Brazil. These authors suggested that fights are very rare or totally absent among males of the species, and a low frequency of copulation point out that the species fits within a mating system called serial monogamy. According

4 Monzón F. and Corcuera J. (1990) Análisis preliminar de madurez sexual en machos de delfín franciscana (Necochea, Provincia de Buenos Aires, Argentina). In 7th Workshop of Specialists in Aquatic Mammals of South America, Valdivia, Chile, p 46. 5 Corcuera, J., 1996. Edad de madurez sexual del delfín Franciscana en el Sur de la Provincia de Buenos Aires Argentina. Abstracts of the VII Reunión de Trabajo de Especialistas en Mamíferos Acuáticos de América del Sur, ˜ del Mar, Chile, p. 39. Vina

Author's personal copy 48

M.V. Panebianco et al. / Animal Reproduction Science 131 (2012) 41–48

to this system, a male and a female mate during a reproductive cycle, being then the female able to mate with a different male (or not) in successive reproductive cycles, as suggested by Danilewicz et al. (2004). 5. Conclusions The present study accomplishes to provide new information about reproductive aspects of male southernmost population of P. blainvillei. Based on the ASM and LSM values obtained will be possible to assess trends in this population affected by accidental mortality. Furthermore, provides a new analytical tool, the hierarchical cluster analysis, to determine the sexual maturity based on gonad and body morphometricals measures. Acknowledgments This study is part of the PhD thesis of MV Panebianco presented at University of Buenos Aires (UBA). Our sincere gratitude to all artisanal fishermen whom made this project possible. The authors would like to thank to D. Rodriguez and P. Denuncio for the gonads sample given, to Daniel Danilewicz for helpful comments and general support. We are in debt with K. Arias and L. Nogueira (MACN/CONICET, EHPQ) as well as to M. Sotelo, and M. V. Massola (RUM Bahía Blanca, Bahía Falsa y Bahía Verde) for their continuous collaboration during the sampling survey. P. Penchaszadeh (CONICET, MACN/CONICET; FCEyN, UBA), P. Faillace and R. Bernabeu (FM, UBA/CONICET), M.N. Paso-Viola (LECyMM, MACN/CONICET), F.H. Pérez (Ecocentro) and G.J. Miano, thanks for their support. We are grateful with Féliz Azára Foundation and A. Giacchino, for their financial support. The project was funded by grants from University of Valencia (HLC), the Society of Marine Mammalogy and the Cetacean Society International (MVP). References American Society of Mammalogists, 1961. Standardized methods for measuring and recording data on the smaller cetaceans. J. Mammal. 42, 471–476. Bertozzi, C., 2009. Interac¸ão com a pesca: implicac¸oes na conservac¸ão na toninha, Pontoporia blainvillei (Cetacea, Pontoporiidae) no litorial no estado de São Pablo, SP. PhD thesis. Instituto Oceanográfico na Universidade de São Paulo, São Paulo, Brazil. Bordino, P., Albareda, D., 2004. Incidental mortality of Franciscana dolphin Pontoporia blainvillei in coastal gillnet fisheries in northern Buenos Aires, Argentina. Report of the International Whaling Commission Meeting, Sorrento, Italia, N◦ SC/56/SM11. Botta, S., 2005. Reproduc¸ão e crescimento dos machos de toninha (Pontoporia blainvillei) do Rio Grande do Sul, Brasil. MSc thesis. Universidade Federal do Rio Grande, Rio Grande, Brazil. Brownell Jr., R.L., 1989. Franciscana Pontoporia blainvillei (Gervais & dˇıOrbigny, 1844). In: Ridgway, S.H., Harrison, R. (Eds.), Handbook of

Marine Mammals: River Dolphins and the Larger Toothed Whales, vol. 4. Academic Press, San Diego, p. 442. Cappozzo, H.L., Negri, M.F., Pérez, F.H., Albareda, D., Monzón, F., Corcuera, J.F., 2007. Incidental mortality of franciscana dolphin, (Pontoporia blainvillei), in Argentina. LAJAM 6, 127–137. Caswell, H., Brault, S., Read, A.J., Smith, T.D., 1998. Harbor porpoise and fisheries: an uncertainty analysis of incidental mortality. Ecol. Appl. 8, 1226–1238. Corcuera, J., Monzón, F., Crespo, E.A., Aguilar, A., Raga, J.A., 1994. Interactions between marine mammals and the coastal fisheries of Necochea and Claromecó (Buenos Aires Province, Argentina). Rep. Int. Whaling Commission (Special issue 15), 283–290. Crespo, E.A., Harris, G., González, R., 1998. Group size and distributional range of the Franciscana Pontoporia blainvillei. Mar. Mamm. Sci. 14, 845–849. Danilewicz, D., 2003. Reproduction of female franciscana (Pontoporia blainvillei) in Rio Grande do Sul, Southern Brazil. LAJAM 2, 67–78. Danilewicz, D., Clave r, J.A., Perez-Carrera, A.L., Secchi, E.R., Fontoura, N.F., 2004. Reproductive biology of male franciscanas (Pontoporia blainvillei) (Mammalia, Cetacea) from Rio Grande do Sul, Southern Brazil. Fish. Bull. 102, 581–592. DeMaster, D.P., 1978. Calculation of the average age of sexual maturity in marine mammals. J. Fish. Res. Board Can. 35, 912–915. Di Rienzo, J.A., Casanoves, F., Balzarini, M.G., Gonzales, L., Tablada, M., Robledo, C.W., 2010. InfoStat versión 2010. Grupo InfoStat, FCA, Universisdad Nacional de Córdoba, Argentina. Ferrero, R.C., Walker, W.A., 1999. Age, growth and reproductive patterns of Dallˇıs porpoise (Phocoenoides dalli) in the central North Pacific Ocean. Mar. Mamm. Sci. 15, 273–313. Harrison, R.J., Brownnell Jr., R.L., 1971. The gonads of the South American dolphins, Inia geoffrensis, Pontoporia blainvillei, and Sotalia fluviatilis. J. Mamma. 52, 413–419. Harrison, R.J., Bryden, M.M., McBrearty, D.A., Brownell Jr., R.L., 1981. The ovaries and reproduction in Pontoporia blainvillei (Cetacea: Platanistidae). J. Zool. 193, 563–580. Hohn, A.A., Chiver, S.J., Barlow, J., 1985. Reproductive maturity and seasonality of male spotted dolphins, Stenella attenuata, in the eastern tropical pacific. Mar. Mamm. Sci. 1, 273–293. IUCN (2010) IUCN Red List of Threatened Species. Version 2010.4. Available from http://www.iucnredlist.org/apps/redlist/details/17978/0 [accessed 18 October 2011]. Kasuya, T., Brownell Jr., R.L., 1979. Age determination, reproduction and growth of the franciscana dolphin, Pontoporia blainvillei. Sci. Rep. Whales Res. Inst. 31, 43–67. Negri. M.F., 2010. Estudio de la biología y ecología del delfín franciscana, Pontoporia blainvillei, y su interacción con la pesquería costera en la provincia de Buenos Aires. PhD thesis. Universidad de Buenos Aires, Buenos Aires, Argentina. Pinedo, M.C., 1991. Development and variation off the franciscana Pontoporia blainvillei. PhD thesis. University of California, Santa Cruz, USA. Pinedo, M.C., Hohn, A.A., 2000. Growth layer patterns in teeth from the franciscana, Pontoporia blainvillei: developing a model for precision in age estimation. Mar. Mamm. Sci. 16, 1–27. Ramos, R.M.A., Di Benedito, A.P.M., Lima, N.R.W., 2000. Growth parameters of Pontoporia blainvillei (Cetácea) in northern Rio de Janeiro. Brazil. Aquat. Mamm. 26, 65–75. Rosas, F.C., Monteiro-Filho, E.L.A., 2001. Reproductive parameters of Pontoporia blainvillei (cetacean, Pontoporiidae), on the coast of Sao Paulo and Paraná States, Brazil. Mammalia 2, 231–245. Secchi, E.R., Wang, J.Y., Murray, B.W., Rocha-Campos, C.C., White, B.N., 1998. Population differentiation in the franciscana (Pontoporia blainvillei) from two geographic locations in Brazil as determined from mitochondrial DNA control region sequences. Can. J. Zool. 76, 1622–1627. Siciliano, S., 1994. Review of small cetaceans and fishery interactions in coastal waters of Brazil. Rep. Int. Whaling Commission (Special issue 15), 241–250.