J Med Primatol doi:10.1111/jmp.12111
ORIGINAL ARTICLE
Semen characteristics of captive common marmoset (Callithrix jacchus): a comparison of a German with a Brazilian colony Augusto Rodrigo del Rio do Valle1,2,3, Cristiane Macedo del Rio do Valle3, Marcilio Nichi4, Jose ~es4 Pereira Carneiro Muniz5, Penelope Lea Nayudu6 & Marcelo Alcindo de Barros Vaz Guimara 1 2 3 4 5 6
Institute of Health Sciences, Paulista University, S~ ao Paulo, Brazil Applied Research Division, S~ ao Paulo Zoological Park Foundation, S~ao Paulo, Brazil Wildlife Management Consultoria Veterin aria, S~ ao Paulo, Brazil Animal Reproduction Department, University of S~ ao Paulo, S~ao Paulo, Brazil National Primate Center, Health Ministry, Ananindeua, Brazil Reproductive Biology Unit, German Primate Center, Goettingen, Germany
Keywords acrosome – plasma membrane – primate – PVS – sperm – vibrostimulation Correspondence Rodrigo del Rio do Valle, Rua Miguel Stefano, 4241, S~ ao Paulo, SP CEP 04301-905, Brazil. Tel.: +55 11 98265 4080; fax: +55 11 3768 2621; e-mail: [email protected] Accepted February 20, 2014.
Abstract Background The common marmoset (Callithrix jacchus) is of interest for reproductive studies because of their high and variable fertility in captivity. However, to understand this variability, much basic information still needs to be gathered. This study is the first to characterize marmoset semen comparing a German and a Brazilian colony. Methods All animals were weighted, and semen samples were collected by penile vibratory stimulation and analyzed according to standard criteria. Results and Conclusions The German animals had a higher mean and maximum weight than the Brazilian. Further, sperm concentration tended to be higher with heavier animals in the German population, but not in the Brazilian. Additionally, major defects tended to be more frequent with higher weight but also only in German sample. In spite of these differences, the total range and variability in sperm concentration were similar in both populations, suggesting that this is characteristic for the species.
Introduction The common marmoset (Callithrix jacchus) is widely used in biomedical and applied research, in particular reproductive studies [4, 7, 8, 10, 11, 13, 17]. Further, because of its easy availability and non-endangered status, this species is already being used as a model for threatened closely related species in Brazil. The development of reproductive technologies, such as sperm banking as tools for genetic management, is an important goal for which common marmoset can be a key model. Knowledge of semen characteristics and sperm quality for each species are critical to the development of effective assisted reproductive strategies, therefore must be established for each captive colony and for free-living populations. Special attention must also be given to J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
optimized semen collection methods and cryostorage as well as the effects of different aspects of captive husbandry [6, 9]. Although, to our knowledge, captive management has been developed empirically and largely separately for each colony, a general phenomenon of increased mean body weight and variability of weight as well as an increase in mean litter size is common knowledge for captive marmosets and has been already published [15]. Furthermore, as European and North American captive colonies have been genetically isolated from the wild populations for around half a century, an ample time span has been available for selective pressures of the captive environments to have their progressive effects without dilution effects by new animals from the wild. 225
Common marmoset semen characteristics two colonies
Our group has made a study of the association of high weight and chronic diseases in the DPZ colony [16], which will be published soon. This association has been well documented in humans [5] and has also been published for marmosets in USA [12]. We have collected extensive data on the effects of high body weight on female reproductive function, but to date, no study has been carried out investigating the relationship of body weight and semen characteristics in marmoset. The aim of this study was therefore to provide initial data on this issue and establish some basic semen parameters for the species, using samples from an European colony with a high mean body weight and genetically isolated from the wild, compared with a Brazilian colony with a lower mean body weight and not genetically isolated from the wild. Materials and methods Humane care guidelines All procedures were performed in accordance with the German animal protection law (Animal Experiment Permission No. AZ509.42502/08-01.03) and the Brazilian animal protection law (Ethical Committee for the Use of Animals in Research, Animal Experiment Permission No. 489/2004; IBAMA Permission 39/2005). Animal selection and housing conditions Common marmosets used in this study were randomly selected from the males available in each colony, aged between 2 and 8 years. Populations compared were housed at the German Primate Center (DPZ), G€ ottingen, Germany (eight individuals), and the National Primate Center (CENP), Ananindeua, Brazil (six individuals). In the DPZ colony, marmosets were housed in indoor cages with no visual contact between different pairs. The temperature (25 3°C), humidity (55 10%), and light cycle (12-hour light, 12-hour dark) were controlled. Water was available ad libitum, and food was supplied twice daily and was based on commercial aten GmbH, pellets for primates (ssniffâ, ssniff Spezialdi€ Soest, Germany) and cats (dominoâ, Kofur Handelsgesellschaft mbH, Hamburg, Germany). A cooked cereal mixture was given for breakfast. And for the main meal, animals were fed cooked meat, a rotating selection of fresh fruits and vegetables supplemented by vitamins, proteins, minerals, and Arabic gummy and occasional meal worms were supplied. This colony has been genetically isolated from the wild for many generations. 226
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In the CENP colony (located in the Amazon region Lat: 1°220 57″ and Long: 48°220 52″), marmosets were also held in indoor cages with no visual contact between different pairs, but had access to natural light and air. The diet was based on assorted tropical fruits and vegetables offered daily, and commercial pellets for dogs (choice based on 28% crude protein, no specific company) and water were available ad libitum. Commercially available powdered cow’s milk, and vitamins and minerals were given diluted in water thrice weekly. This colony was genetically open, with occasional inclusion of new animals from the wild. Experimental design of semen collection and dilution The analysis of fresh semen samples presented here was performed retrospectively. The samples were collected as part of a study aimed at optimizing cryopreservation methods for this species. Fresh semen samples were collected with a minimum interval of 7 days between sampling. The original semen samples were evaluated fresh for the evaluation of reproductive status of the animals, and the rest of the sample was used for cryopreservation experiments (results to be published separately). In both colonies, males were separated from respective females 12–14 hours prior to sperm collection, but remained in visual contact. With the exception of two animals from the DPZ colony, animals had never before been used for semen collection. Semen samples were collected by penile vibratory stimulation (PVS) using a vibratory apparatus (FertiCareâ personal, Multicept ApS, Rungsted, Denmark), according to protocol [17] developed and published from our group. The semen was collected into a specially made clean (sterile) dry glass tube and immediately diluted with 100 ll of modified TALP–HEPES medium (TALP-HEPES + 3 mg/ml BSA V, 0.25 mM Na pyruvate pH 7.33) at 37°C [17]. Semen evaluation Samples in both colonies were evaluated according to a standard set of parameters by the same experienced investigator. Physical characteristics of ejaculates were analyzed immediately after ejaculation. The pH was evaluated by placing a drop of semen on a pH strip (Merckâ, Darmstadt, Germany). Volume was measured using a positive-displacement pipette (Gilsonâ, Middleton, WI, USA) after dilution with 100 ll of medium. Sperm motility and forward progressive sperm motility were evaluated at 37°C with a phase-contrast microscope using a magnification of 2009. The integrity of the sperm plasmatic membrane was accessed with the eosin–nigrosin stain technique previJ Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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ously validated for this species [17]. Duplicate smears from each sample were analyzed, and 200 sperm were examined at a magnification of 4009. Acrosomal status was evaluated using the simple staining technique for acrosome status according to protocol validated for common marmosets sperm [17]. Duplicate smears were prepared on microscope glass slides, and 200 sperm from each sample were evaluated at a magnification of 10009 under oil immersion. To determine sperm concentration, 5 ll of diluted semen was added to 45 ll of 10% formalin saline solution and counted in a hemocytometer [3]. For morphology evaluation, duplicate smears of diluted semen were prepared on glass slides and stained with SpermOscanâ (NidaCon, Moelndal, Switzerland), following the manufacturer instructions. In brief, slides were submerged five times in each of the three stain solutions and air-dried. Subsequently, the slides were mounted with Eukittâ (Fluka BioChemika, Buchs SG, Switzerland) and coverslipped to make a permanent preparation. After drying overnight, 200 sperm from each sample were evaluated for morphology [2] at a magnification of 10009. Statistical analysis All data were evaluated using SAS System for Windows (SAS Institute Inc., Cary, NC, USA). The mean value and range for all seminal parameters evaluated were calculated for each colony separately and for both colonies together. Also, paired analyses were carried out. The effect of colony (CENP vs. DPZ) was determined using parametric (Student’s t-test) and nonparametric (Mann–Whitney U-test) tests, according to the residue normality (Gaussian distribution) and variance homogeneity of each variable. The Spearman correlation coefficient analysis was used to determine whether there was a relationship between all seminal parameters and weight. The Fisher’s exact test was performed to analyze the variability of sperm concentration related to weight to determine whether there was a difference in the distribution between the two colonies. A probability value of P < 0.05 was considered statistically significant. Results are reported as untransformed means, standard error, the range (minimum and maximum values), and correlation coefficient (r).
Common marmoset semen characteristics two colonies
Student’s t-test (P = 0.078), the mean of the body mass for German animals was approximately 16% higher than for Brazilian (German = 392.9 g, SE = 18.1, range = 145; Brazilian = 336.7 g, SE = 23.55, range = 160). Figure 1 shows that this difference is based on a higher maximum weight for the German group, with the heaviest animal from Brazil below the mean value from the German sample. Weights from each animal are presented in a crescent order to show the weight variability comparing the Brazilian and the German samples. Red line is the mean value for the German samples. Although there was no significant difference between colonies in the range of seminal characteristics when weight was not considered (Table 1), analysis of sperm concentration distribution related to body mass using Fisher’s exact test revealed higher sperm concentrations in samples from the German animals weighing around and over the mean body mass (P = 0.007) (Fig. 2A). In contrast, the same analysis showed no relationship of sperm concentration to body mass in the Brazilian group (Fig. 2B). The distribution of the samples is shown based on the correlation between sperm concentration and weight in the German (A) and Brazilian (B) samples. In (A), the heaviest animals presented the highest sperm concentration. (A) and (B): The red line shows the mean value for sperm concentration, and the blue line shows the mean value for body mass. The Spearman correlation coefficient analysis showed a positive correlation between major sperm defects and weight (P = 0.02, r = 0.4080) with both colonies analyzed together, but this association was primarily due to the highest weight German animals as shown in Figure 3. The correlation between sperm major defects and weight is presented for the Brazilian and German samples showing from German samples the heaviest animals had the highest proportion of sperm major defects.
Results Sperm collection attempts with animals that had never been used for this purpose by any artificial technique resulted in 83.33% of success in obtaining semen. Although the animals in the two colonies did not differed significantly in body mass evaluated by J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Fig. 1 Weight variability comparing Brazilian and German samples.
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Table 1 Seminal parameters from two different colonies of common marmosets (Callithrix jacchus), Centro Nacional de Primatas—CENP in Brazil and Deutsches Primatenzentrum—DPZ in Germany, collected by penile vibrostimulation Colony CENP (n = 19) Mean SE (range) Volume (µl) pH Motility (%) Forward progressive motility (%) Sperm plasmatic membrane integrity (%) Acrosome integrity (%) Concentration (x 106 spz/ml) Normal sperm (%) Major defects (%) Minor defects (%)
DPZ (n = 22) Mean SE (range)
26.05 7.6 83.7 77.9 84.2
2.88 (5–55) 0.04 (7.4–8.0) 2.19 (65–95) 2.38 (60–90) 2.08 (63–97)
84.1 1096.99 63.6 16.9 19.5
2 (68.5–96) 85.1 3.93 (33.5–100) 252.37 (36.18–3557.3) 1032.88 205.56 (38.7–3375) 3.41 (39–85) 61.7 4.74 (26–80) 2.05 (2.5–31) 15.9 1.71 (9.5–27.5) 2.05 (7.5–37) 22.5 4 (7.5–37) P > 0.05
27.05 7.6 81.8 75.0 85.3
CENP + DPZ (n = 41) Mean SE (range)
35.0
(A)
26.59 7.6 82.7 76.3 84.7
1.85 (5–55) 0.03 (7.4–8.0) 1.98 (20–95) 2.06 (20–90) 2.05 (21–98)
84.6 1062.59 62.8 16.5 20.7
2.28 (33.5–100) 158.79 (36.18–3557.3) 2.75 (26–85) 1.39 (2.5–31) 2.01 (7.5–59.5)
Germany Brazil
30.0
Sperm Major defects (%)
2.45 (5–50) 0.05 (7.4–8.0) 3.2 (20–90) 3.27 (20–90) 3.42 (21–98)
25.0 20.0 15.0 10.0 5.0
(B)
0.0
200
250
300
350
400
450
500
Weight (g) Fig. 3 Sperm major defects vs. weight comparing Brazilian and German samples.
Discussion
Fig. 2 (A) Sperm concentration vs. weight in Germany. (B) Sperm concentration vs weight in Brazil.
The Spearman correlation coefficient analysis also showed a positive correlation between acrosome status and motility in Brazilian ejaculates (P = 0.004, r = 0.62524), but not for the German samples. 228
The present study compares, for the first time, Callithrix jacchus semen characteristics between a random sample of adult males in two colonies (in Germany and Brazil) under controlled conditions using penile vibratory stimulation (PVS), which has been shown to be the most effective technique for Callithrix jacchus sperm collection [7, 11, 14]. Sperm collection by PVS is an already established method for Callithrix jacchus [11, 13, 16] in captivity. Our high rate of success with animals having no previous experience with vibrostimulation (i.e., success rate of obtaining a first sample), similar to that previously reported by Schneiders et al. [14], strongly suggests that this method would be applicable to wild individuals. J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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This method also produces a larger and better quality sample than that previously reported by electroejaculation and is more convenient and controllable than the vaginal wash strategy reported by Morrel et al. [11]. Both colonies evaluated in this study showed a similar range of all semen variables, leading us to conclude that this variability was characteristic for the species in captivity. However, as the two colonies differed in major ways in the captive management and environment, and the distribution and mean body mass of the males in the two colonies differed although not significantly, some associated differences in semen characteristics might have been predicted. And indeed, an analysis of the distribution of body weight related to sperm concentration revealed different patterns in each colony. Our study group from the German colony demonstrated a significantly higher proportion of samples with sperm concentrations greater than the mean were from the heaviest weight animals (defined as animals above the mean weight for the colony, >392.9 g). Animals with body mass below 380 g had consistently below mean sperm concentrations. In contrast, the Brazilian colony, which had the same weight range as the lighter German animals, produced both high and low sample counts. One possible explanation of this difference may lay with the captive management. In a captive environment which promotes weight gain and adiposity such as in the German colony, those animals which do not reach high weights or which are losing weight may have a suboptimal health status, and this may also have a negative influence on sperm production. Even though the mean weight of the Brazilian animals was lower than the German group, the heaviest did exceed the reported weights of wild animals [1], so it could be assumed that changes due to captivity had already begun to occur in this group. However, regular additions to the colony from wild individuals presumably assist in moderating the changes in body mass. This contrasts with the German colony which has been completely isolated from the wild for nearly half a century and living under conditions which differ drastically from those which occur in the wild. In relation to the actual technique of collection and analysis methods, the present study produced a higher proportion of sperm with intact plasma membrane than the two previous reports [7, 14] using the PVS collection method. This difference could be related to the improvements in semen handling or the accuracy of the validated evaluation methods [17] used in the present study. The
J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Common marmoset semen characteristics two colonies
total number of motile sperm was also higher with the present study than for Kuederling et al. [7], and double of the value reported by Schneiders et al. [14]. Further differences were found in sperm morphology from previous reports using PVS. Schneiders et al. [14] reported a higher percentage of normal sperm than results presented here. A possible reason for this difference could be differences in the evaluation methods. In our study, a standard method with commercial kits for human sperm analysis was used. It was therefore necessary to prepare smears of the samples, a procedure not needed with the use of a CASA system used by Schneiders et al. [14]. Although a CASA system was not used for the analysis in this study, the critical and accurate analysis performed in our study, including analysis of additional parameters not presented in these previous studies, such as integrity of acrosome membrane (a method validated for marmoset by us), rectilinear progressive motility, and a detailed analysis of the morphology produced very stabile and reliable results. We further showed that PVS can be effectively used without any previous training of the animals. The results using these methods showed that the range of sample qualities was similar between two very different colonies and can therefore be assumed to typical of captive marmosets. Additionally, the increased high end weights of the German colony suggested that weight-associated physiologic differences may have an effect on spermatogenesis. This study introduces a systematic simple set of methods to collect high-quality semen samples from naive animals. The results represent the first information of its kind on this topic and provide a technical and informational basis on which to base further studies and to assist development of sperm banking for related primate species. Acknowledgments We would like to thank the German Primate Center (DPZ, Deutsches Primatenzentrum, Goettingen, Germany) and the National Primate Center (CENP, Centro Nacional de Primatas, Ananindeua, Brazil) for the facilities and access to the animals. The first author would also like to thank the National Council for Scientific and Technological Development (CNPq, Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico, Brazil) and the German Academic Exchange Service (DAAD, Deutscher Akademischer Austausch Dienst, Germany) for personal financial support and travel costs.
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References 1 Ara ujo A, Sousa MBC: Testicular volume and reproductive status of wild Callithrix jacchus. Int J Primatol 2008; 29:1355–64. 2 Blom E: The ultrastructure of some characteristic sperm defects and a proposal for a new classification of bull spermogram. Nord Vet Med 1973; 25:383–91. 3 Colegio Brasileiro de Reproducß~ao Animal. Manual Para Exame Androl ogico e Avaliacß~ao de S^emen Animal, 2nd edn. Belo Horizonte: Colegio Brasileiro de Reproducß~ao Animal, 1998. 4 Cui KH, Flaherty SP, Newble CD, Guerin MV, Napier AJ, Matthews CD: Collection and analysis of semen from the common marmoset (Callithrix jacchus). J Androl 1991; 12:214–20. 5 Henegar JR, Bigler SA, Henegar LK, Tyagi SC, Hall JE: Functional and structural changes in the kidney in the early stages of obesity. J Am Soc Nephrol 2001; 12:1211–17. 6 Kanakaraj P, Krishnamurthy US: Influence of potassium and sodium ions on the quality of buffalo semen. Cheiron 1984; 13: 48–50. 7 Kuederling I, Schneiders A, Sonksen J, Nayudu PL, Hodges JK: Non-invasive collection of ejaculates from the common marmoset (Callithrix jacchus) using penile
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vibrostimulation. Am J Primatol 2000; 52:149–54. Li LH, Donald JM, Golub MS: Review on testicular development, structure, function, and regulation in common marmoset. Birth Defects Res B 2005; 74:450–69. Meacham TN, Cunha TJ, Warnick AC, Hentges JF Jr, Hargrove DD: Influence of low protein rations on growth and semen characteristics of young beef bulls. J Anim Sci 1963; 22:115–20. Millar MR, Sharpe RM, Weinbauer GF, Fraser HM, Saunders PTK: Marmoset spermatogenesis: organizational similarities to the human. Int J Androl 2000; 23:266–77. Morrel JM, Kuederling I, Hodges JK: Influence of semen collection method on ejaculate characteristics in the common marmoset, Callithrix jacchus. J Androl 1996; 17:164–72. Power ML, Ross CN, Schulkin J, Ziegler TE, Tardif SD: Metabolic consequences of the early onset of obesity in common marmoset monkeys. Obesity (Silver Spring) 2013; 21:E592–8. Pudritz S. Untersuchungen zu Kapazitierung und Akrosomreaktion der Spermatozoen des Weißb€ uschelaffen (Callithrix jacchus) in vitro. [Characterization and regulation of sperm capacitation and acrosome
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reaction in vitro in the marmoset monkey (Callithrix jacchus)]. (PhD thesis) – Faculty of Biology, University of Marburg, Marburg, 2000, pp 115. Schneiders A, Sonksen J, Hodges JK: Penile vibratory stimulation in the marmoset monkey: a practical alternative to electro-ejaculation, yielding ejaculates of enhanced quality. J Med Primatol 2004; 33:98–104. Tardif SD, Jaquish CE: Ovulation number in the marmoset monkey (Callithrix jacchus): relation to body weight, age and repeatability. Am J Primatol 1997; 42:323–9. Valle CMR. Estudo Morfofuncional de rins de primatas Callithrix jacchus em cativeiro. [Morphofunctional study in kidneys of captive Callithrix jacchus primates]. (PhD thesis) – Faculty of Veterinary Medicine and Zootechny, University of S~ao Paulo, S~ao Paulo, 2008, pp 158. Valle RR, Valle CMR, Nichi M, Muniz JAPC, Nayudu PL, Guimar~aes MABV: Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm. Theriogenology 2008; 70:115–20.
J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
ORIGINAL ARTICLE
Semen characteristics of captive common marmoset (Callithrix jacchus): a comparison of a German with a Brazilian colony Augusto Rodrigo del Rio do Valle1,2,3, Cristiane Macedo del Rio do Valle3, Marcilio Nichi4, Jose ~es4 Pereira Carneiro Muniz5, Penelope Lea Nayudu6 & Marcelo Alcindo de Barros Vaz Guimara 1 2 3 4 5 6
Institute of Health Sciences, Paulista University, S~ ao Paulo, Brazil Applied Research Division, S~ ao Paulo Zoological Park Foundation, S~ao Paulo, Brazil Wildlife Management Consultoria Veterin aria, S~ ao Paulo, Brazil Animal Reproduction Department, University of S~ ao Paulo, S~ao Paulo, Brazil National Primate Center, Health Ministry, Ananindeua, Brazil Reproductive Biology Unit, German Primate Center, Goettingen, Germany
Keywords acrosome – plasma membrane – primate – PVS – sperm – vibrostimulation Correspondence Rodrigo del Rio do Valle, Rua Miguel Stefano, 4241, S~ ao Paulo, SP CEP 04301-905, Brazil. Tel.: +55 11 98265 4080; fax: +55 11 3768 2621; e-mail: [email protected] Accepted February 20, 2014.
Abstract Background The common marmoset (Callithrix jacchus) is of interest for reproductive studies because of their high and variable fertility in captivity. However, to understand this variability, much basic information still needs to be gathered. This study is the first to characterize marmoset semen comparing a German and a Brazilian colony. Methods All animals were weighted, and semen samples were collected by penile vibratory stimulation and analyzed according to standard criteria. Results and Conclusions The German animals had a higher mean and maximum weight than the Brazilian. Further, sperm concentration tended to be higher with heavier animals in the German population, but not in the Brazilian. Additionally, major defects tended to be more frequent with higher weight but also only in German sample. In spite of these differences, the total range and variability in sperm concentration were similar in both populations, suggesting that this is characteristic for the species.
Introduction The common marmoset (Callithrix jacchus) is widely used in biomedical and applied research, in particular reproductive studies [4, 7, 8, 10, 11, 13, 17]. Further, because of its easy availability and non-endangered status, this species is already being used as a model for threatened closely related species in Brazil. The development of reproductive technologies, such as sperm banking as tools for genetic management, is an important goal for which common marmoset can be a key model. Knowledge of semen characteristics and sperm quality for each species are critical to the development of effective assisted reproductive strategies, therefore must be established for each captive colony and for free-living populations. Special attention must also be given to J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
optimized semen collection methods and cryostorage as well as the effects of different aspects of captive husbandry [6, 9]. Although, to our knowledge, captive management has been developed empirically and largely separately for each colony, a general phenomenon of increased mean body weight and variability of weight as well as an increase in mean litter size is common knowledge for captive marmosets and has been already published [15]. Furthermore, as European and North American captive colonies have been genetically isolated from the wild populations for around half a century, an ample time span has been available for selective pressures of the captive environments to have their progressive effects without dilution effects by new animals from the wild. 225
Common marmoset semen characteristics two colonies
Our group has made a study of the association of high weight and chronic diseases in the DPZ colony [16], which will be published soon. This association has been well documented in humans [5] and has also been published for marmosets in USA [12]. We have collected extensive data on the effects of high body weight on female reproductive function, but to date, no study has been carried out investigating the relationship of body weight and semen characteristics in marmoset. The aim of this study was therefore to provide initial data on this issue and establish some basic semen parameters for the species, using samples from an European colony with a high mean body weight and genetically isolated from the wild, compared with a Brazilian colony with a lower mean body weight and not genetically isolated from the wild. Materials and methods Humane care guidelines All procedures were performed in accordance with the German animal protection law (Animal Experiment Permission No. AZ509.42502/08-01.03) and the Brazilian animal protection law (Ethical Committee for the Use of Animals in Research, Animal Experiment Permission No. 489/2004; IBAMA Permission 39/2005). Animal selection and housing conditions Common marmosets used in this study were randomly selected from the males available in each colony, aged between 2 and 8 years. Populations compared were housed at the German Primate Center (DPZ), G€ ottingen, Germany (eight individuals), and the National Primate Center (CENP), Ananindeua, Brazil (six individuals). In the DPZ colony, marmosets were housed in indoor cages with no visual contact between different pairs. The temperature (25 3°C), humidity (55 10%), and light cycle (12-hour light, 12-hour dark) were controlled. Water was available ad libitum, and food was supplied twice daily and was based on commercial aten GmbH, pellets for primates (ssniffâ, ssniff Spezialdi€ Soest, Germany) and cats (dominoâ, Kofur Handelsgesellschaft mbH, Hamburg, Germany). A cooked cereal mixture was given for breakfast. And for the main meal, animals were fed cooked meat, a rotating selection of fresh fruits and vegetables supplemented by vitamins, proteins, minerals, and Arabic gummy and occasional meal worms were supplied. This colony has been genetically isolated from the wild for many generations. 226
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In the CENP colony (located in the Amazon region Lat: 1°220 57″ and Long: 48°220 52″), marmosets were also held in indoor cages with no visual contact between different pairs, but had access to natural light and air. The diet was based on assorted tropical fruits and vegetables offered daily, and commercial pellets for dogs (choice based on 28% crude protein, no specific company) and water were available ad libitum. Commercially available powdered cow’s milk, and vitamins and minerals were given diluted in water thrice weekly. This colony was genetically open, with occasional inclusion of new animals from the wild. Experimental design of semen collection and dilution The analysis of fresh semen samples presented here was performed retrospectively. The samples were collected as part of a study aimed at optimizing cryopreservation methods for this species. Fresh semen samples were collected with a minimum interval of 7 days between sampling. The original semen samples were evaluated fresh for the evaluation of reproductive status of the animals, and the rest of the sample was used for cryopreservation experiments (results to be published separately). In both colonies, males were separated from respective females 12–14 hours prior to sperm collection, but remained in visual contact. With the exception of two animals from the DPZ colony, animals had never before been used for semen collection. Semen samples were collected by penile vibratory stimulation (PVS) using a vibratory apparatus (FertiCareâ personal, Multicept ApS, Rungsted, Denmark), according to protocol [17] developed and published from our group. The semen was collected into a specially made clean (sterile) dry glass tube and immediately diluted with 100 ll of modified TALP–HEPES medium (TALP-HEPES + 3 mg/ml BSA V, 0.25 mM Na pyruvate pH 7.33) at 37°C [17]. Semen evaluation Samples in both colonies were evaluated according to a standard set of parameters by the same experienced investigator. Physical characteristics of ejaculates were analyzed immediately after ejaculation. The pH was evaluated by placing a drop of semen on a pH strip (Merckâ, Darmstadt, Germany). Volume was measured using a positive-displacement pipette (Gilsonâ, Middleton, WI, USA) after dilution with 100 ll of medium. Sperm motility and forward progressive sperm motility were evaluated at 37°C with a phase-contrast microscope using a magnification of 2009. The integrity of the sperm plasmatic membrane was accessed with the eosin–nigrosin stain technique previJ Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
del Rio do Valle et al.
ously validated for this species [17]. Duplicate smears from each sample were analyzed, and 200 sperm were examined at a magnification of 4009. Acrosomal status was evaluated using the simple staining technique for acrosome status according to protocol validated for common marmosets sperm [17]. Duplicate smears were prepared on microscope glass slides, and 200 sperm from each sample were evaluated at a magnification of 10009 under oil immersion. To determine sperm concentration, 5 ll of diluted semen was added to 45 ll of 10% formalin saline solution and counted in a hemocytometer [3]. For morphology evaluation, duplicate smears of diluted semen were prepared on glass slides and stained with SpermOscanâ (NidaCon, Moelndal, Switzerland), following the manufacturer instructions. In brief, slides were submerged five times in each of the three stain solutions and air-dried. Subsequently, the slides were mounted with Eukittâ (Fluka BioChemika, Buchs SG, Switzerland) and coverslipped to make a permanent preparation. After drying overnight, 200 sperm from each sample were evaluated for morphology [2] at a magnification of 10009. Statistical analysis All data were evaluated using SAS System for Windows (SAS Institute Inc., Cary, NC, USA). The mean value and range for all seminal parameters evaluated were calculated for each colony separately and for both colonies together. Also, paired analyses were carried out. The effect of colony (CENP vs. DPZ) was determined using parametric (Student’s t-test) and nonparametric (Mann–Whitney U-test) tests, according to the residue normality (Gaussian distribution) and variance homogeneity of each variable. The Spearman correlation coefficient analysis was used to determine whether there was a relationship between all seminal parameters and weight. The Fisher’s exact test was performed to analyze the variability of sperm concentration related to weight to determine whether there was a difference in the distribution between the two colonies. A probability value of P < 0.05 was considered statistically significant. Results are reported as untransformed means, standard error, the range (minimum and maximum values), and correlation coefficient (r).
Common marmoset semen characteristics two colonies
Student’s t-test (P = 0.078), the mean of the body mass for German animals was approximately 16% higher than for Brazilian (German = 392.9 g, SE = 18.1, range = 145; Brazilian = 336.7 g, SE = 23.55, range = 160). Figure 1 shows that this difference is based on a higher maximum weight for the German group, with the heaviest animal from Brazil below the mean value from the German sample. Weights from each animal are presented in a crescent order to show the weight variability comparing the Brazilian and the German samples. Red line is the mean value for the German samples. Although there was no significant difference between colonies in the range of seminal characteristics when weight was not considered (Table 1), analysis of sperm concentration distribution related to body mass using Fisher’s exact test revealed higher sperm concentrations in samples from the German animals weighing around and over the mean body mass (P = 0.007) (Fig. 2A). In contrast, the same analysis showed no relationship of sperm concentration to body mass in the Brazilian group (Fig. 2B). The distribution of the samples is shown based on the correlation between sperm concentration and weight in the German (A) and Brazilian (B) samples. In (A), the heaviest animals presented the highest sperm concentration. (A) and (B): The red line shows the mean value for sperm concentration, and the blue line shows the mean value for body mass. The Spearman correlation coefficient analysis showed a positive correlation between major sperm defects and weight (P = 0.02, r = 0.4080) with both colonies analyzed together, but this association was primarily due to the highest weight German animals as shown in Figure 3. The correlation between sperm major defects and weight is presented for the Brazilian and German samples showing from German samples the heaviest animals had the highest proportion of sperm major defects.
Results Sperm collection attempts with animals that had never been used for this purpose by any artificial technique resulted in 83.33% of success in obtaining semen. Although the animals in the two colonies did not differed significantly in body mass evaluated by J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Fig. 1 Weight variability comparing Brazilian and German samples.
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Table 1 Seminal parameters from two different colonies of common marmosets (Callithrix jacchus), Centro Nacional de Primatas—CENP in Brazil and Deutsches Primatenzentrum—DPZ in Germany, collected by penile vibrostimulation Colony CENP (n = 19) Mean SE (range) Volume (µl) pH Motility (%) Forward progressive motility (%) Sperm plasmatic membrane integrity (%) Acrosome integrity (%) Concentration (x 106 spz/ml) Normal sperm (%) Major defects (%) Minor defects (%)
DPZ (n = 22) Mean SE (range)
26.05 7.6 83.7 77.9 84.2
2.88 (5–55) 0.04 (7.4–8.0) 2.19 (65–95) 2.38 (60–90) 2.08 (63–97)
84.1 1096.99 63.6 16.9 19.5
2 (68.5–96) 85.1 3.93 (33.5–100) 252.37 (36.18–3557.3) 1032.88 205.56 (38.7–3375) 3.41 (39–85) 61.7 4.74 (26–80) 2.05 (2.5–31) 15.9 1.71 (9.5–27.5) 2.05 (7.5–37) 22.5 4 (7.5–37) P > 0.05
27.05 7.6 81.8 75.0 85.3
CENP + DPZ (n = 41) Mean SE (range)
35.0
(A)
26.59 7.6 82.7 76.3 84.7
1.85 (5–55) 0.03 (7.4–8.0) 1.98 (20–95) 2.06 (20–90) 2.05 (21–98)
84.6 1062.59 62.8 16.5 20.7
2.28 (33.5–100) 158.79 (36.18–3557.3) 2.75 (26–85) 1.39 (2.5–31) 2.01 (7.5–59.5)
Germany Brazil
30.0
Sperm Major defects (%)
2.45 (5–50) 0.05 (7.4–8.0) 3.2 (20–90) 3.27 (20–90) 3.42 (21–98)
25.0 20.0 15.0 10.0 5.0
(B)
0.0
200
250
300
350
400
450
500
Weight (g) Fig. 3 Sperm major defects vs. weight comparing Brazilian and German samples.
Discussion
Fig. 2 (A) Sperm concentration vs. weight in Germany. (B) Sperm concentration vs weight in Brazil.
The Spearman correlation coefficient analysis also showed a positive correlation between acrosome status and motility in Brazilian ejaculates (P = 0.004, r = 0.62524), but not for the German samples. 228
The present study compares, for the first time, Callithrix jacchus semen characteristics between a random sample of adult males in two colonies (in Germany and Brazil) under controlled conditions using penile vibratory stimulation (PVS), which has been shown to be the most effective technique for Callithrix jacchus sperm collection [7, 11, 14]. Sperm collection by PVS is an already established method for Callithrix jacchus [11, 13, 16] in captivity. Our high rate of success with animals having no previous experience with vibrostimulation (i.e., success rate of obtaining a first sample), similar to that previously reported by Schneiders et al. [14], strongly suggests that this method would be applicable to wild individuals. J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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This method also produces a larger and better quality sample than that previously reported by electroejaculation and is more convenient and controllable than the vaginal wash strategy reported by Morrel et al. [11]. Both colonies evaluated in this study showed a similar range of all semen variables, leading us to conclude that this variability was characteristic for the species in captivity. However, as the two colonies differed in major ways in the captive management and environment, and the distribution and mean body mass of the males in the two colonies differed although not significantly, some associated differences in semen characteristics might have been predicted. And indeed, an analysis of the distribution of body weight related to sperm concentration revealed different patterns in each colony. Our study group from the German colony demonstrated a significantly higher proportion of samples with sperm concentrations greater than the mean were from the heaviest weight animals (defined as animals above the mean weight for the colony, >392.9 g). Animals with body mass below 380 g had consistently below mean sperm concentrations. In contrast, the Brazilian colony, which had the same weight range as the lighter German animals, produced both high and low sample counts. One possible explanation of this difference may lay with the captive management. In a captive environment which promotes weight gain and adiposity such as in the German colony, those animals which do not reach high weights or which are losing weight may have a suboptimal health status, and this may also have a negative influence on sperm production. Even though the mean weight of the Brazilian animals was lower than the German group, the heaviest did exceed the reported weights of wild animals [1], so it could be assumed that changes due to captivity had already begun to occur in this group. However, regular additions to the colony from wild individuals presumably assist in moderating the changes in body mass. This contrasts with the German colony which has been completely isolated from the wild for nearly half a century and living under conditions which differ drastically from those which occur in the wild. In relation to the actual technique of collection and analysis methods, the present study produced a higher proportion of sperm with intact plasma membrane than the two previous reports [7, 14] using the PVS collection method. This difference could be related to the improvements in semen handling or the accuracy of the validated evaluation methods [17] used in the present study. The
J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Common marmoset semen characteristics two colonies
total number of motile sperm was also higher with the present study than for Kuederling et al. [7], and double of the value reported by Schneiders et al. [14]. Further differences were found in sperm morphology from previous reports using PVS. Schneiders et al. [14] reported a higher percentage of normal sperm than results presented here. A possible reason for this difference could be differences in the evaluation methods. In our study, a standard method with commercial kits for human sperm analysis was used. It was therefore necessary to prepare smears of the samples, a procedure not needed with the use of a CASA system used by Schneiders et al. [14]. Although a CASA system was not used for the analysis in this study, the critical and accurate analysis performed in our study, including analysis of additional parameters not presented in these previous studies, such as integrity of acrosome membrane (a method validated for marmoset by us), rectilinear progressive motility, and a detailed analysis of the morphology produced very stabile and reliable results. We further showed that PVS can be effectively used without any previous training of the animals. The results using these methods showed that the range of sample qualities was similar between two very different colonies and can therefore be assumed to typical of captive marmosets. Additionally, the increased high end weights of the German colony suggested that weight-associated physiologic differences may have an effect on spermatogenesis. This study introduces a systematic simple set of methods to collect high-quality semen samples from naive animals. The results represent the first information of its kind on this topic and provide a technical and informational basis on which to base further studies and to assist development of sperm banking for related primate species. Acknowledgments We would like to thank the German Primate Center (DPZ, Deutsches Primatenzentrum, Goettingen, Germany) and the National Primate Center (CENP, Centro Nacional de Primatas, Ananindeua, Brazil) for the facilities and access to the animals. The first author would also like to thank the National Council for Scientific and Technological Development (CNPq, Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico, Brazil) and the German Academic Exchange Service (DAAD, Deutscher Akademischer Austausch Dienst, Germany) for personal financial support and travel costs.
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J Med Primatol 43 (2014) 225–230 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
