Reproductive capacity of male bank voles ( Myodes glareolus S chreber , 1780) - age-dependent changes in functional activity of epididymal sperm

. The influence of age on male bank voles’ reproductive tract development, epididymal sperm quantity and functional activity was investigated. Experiments were carried out on male bank voles aged 1.5 to 15 months (n=10 each in 8 age groups). The developmental stage of the reproductive tract was assessed by the weight of testes, seminal vesicles and coagulation glands. In each age group the number of epididymal sperm and their functional activity were examined. Epididymal sperm functional activity was assessed by motility, viability, maturity, head morphology and integrity of the sperm tail membrane. Ageing males were heavier than pre-pubertal and mature ones. Male age also affected the testes, seminal vesicles and coagulation gland development. The heaviest accessory sex glands were noted in 3-month-old males and the lightest in pre-pubertal (1.5-month-old) and older (12-and 15-month-old) males. Sperm counts were significantly higher in 3-, 4-and 5-month-old males than in pre-pubertal and old males. Generally, adult males aged 3-and 4-months , produced sperm of better functional activity. In conclusion, the best male reproductive capacity is found in bank voles of 3 to 4 months of age.


INTRODUCTION
Interest in the effect of paternal age on physical conditions and reproductive efficiency of progeny has recently increased worldwide.Studies on humans and certain rodents show that among anomalous effects associated with advanced male age are pregnancy losses (Serre & robaire, 1998;Slama et al., 2005), stillbirth (aStolfi et al., 2004) and increased postnatal mortality (Zhu et al., 2008).Mutation frequencies in male genomes also increase with age, which may evoke negative effects on individual health, infertility as well as genetic defects in offspring (for a review, see for example Sloter et al., 2004).In the Brown Norway rat (Rattus norvegicus), ageing leads to considerable damage in the testes and to lower serum testosterone levels due to decreased pituitary hormonal activity, and to decreased sperm production (Wang et al., 1993).In old mice more atrophic testes with degenerated seminiferous tubules have been found (Parkening et al., 1988).
The efficiency of spermatogenesis is a key to male reproductive success.Spermatozoa concentrations as well as their functional activity are parameters that can be used to assess the potential success of the sperm and, consequently, the reproductive success of males.Functional activity can be assessed by spermatozoa viability, motility, maturity, integrity of the sperm tail membrane and head morphology.The study of age-dependent changes in sperm quality of the black-footed ferret (Mustela nigripes) based on only two parameters showed that motile and structurally normal sperm was diminished in older males (Wolf et al., 2000).To our knowledge, there are few published reports assessing the influence of male age on complex measurements of sperm functional activity.Studies on mice have shown that mating with males of 12 months of age and older significantly reduced fertilization and decreased implantation potential and fetal viability (katZ-Jaffe et al. 2013).The influence of age has been also demonstrated in studies on the male mosquito, where the optimum male age for successful insemination of females was found to be 4-8 days (SaWadogo et al. 2013).The aim of the present study was to investigate the effect of age on the reproductive development of bank vole (Myodes glareolus) males.The bank vole is presumably the most common small rodent in Europe.This species has become a research object in behavioural, population, and environmental protection studies and it is also used as a classical model for laboratory experiments.Reproductive biology of bank vole is rather well known, including such aspects as time of male sexual maturity (marchleWSka-koJ, 2000), factors influencing mating behaviour (marchleWSka-koJ et al., 2003) and multiple paternity (ratkieWicZ & borkoWSka, 2000).In the wild, bank voles breed seasonally (clarke, 1981).The reproductive season lasts from the beginning of March to the end of October.Males living in a natural environment reach puberty at about two months of age (buJalSka, 1973).In a laboratory colony mature spermatozoa were observed for the first time in 1.5-month-old bank vole males (krucZek, 1986).However the optimal male age for reproduction is still not well determined.In the present study we examined the effects of age on the quality and functional activity of epididymal sperm of bank vole males using different parameters.

Animals
The bank voles used in the experiments came from the Institute of Environmental Sciences, Jagiellonian University in Kraków.The animals are maintained as outbred stock according to the system described by Green (green, 1966).
Briefly, each generation consists of at least 22 breeding pairs; the male and female in each mating pair do not have common parents or grandparents.This breeding system ensures the heterogeneity of the colony.It could be assumed that animals from such a colony are comparable to rodents living in a natural population.Bank voles were kept in polyethylene cages (36 x 21 x 17 cm) under a 14 h photoperiod (lights on at 06:00 a.m.) at 20±2° C. Standard pelleted chow and water were available ad libitum.Wood shavings were provided as bedding material and changed once a week.The males were housed, at least in 70% of cases, as three per cage from weaning at 19-20 days; only occasionally were there two or four males per cage.

Procedures of body and organ weights and of epididymal sperm evaluation
At the appropriate age, the males were sacrificed by cervical dislocation and weighed.The paired testes and seminal vesicles and coagulating glands were dissected out and weighed (the latter two jointly) too.The procedures and methods to count and observe spermatozoa were the same as used for mice (Styrna & krZanoWSka, 1995;Styrna et al., 2003) as well as for bank voles in our previous paper (krucZek & Styrna, 2009).The individual counting and analyzing of epididymal sperm were conducted without information on the males' ages.

Preparation of epididymal sperm suspension
After gentle pressing of each cauda epididymis with forceps, allowing sperm to pass to the vasa deferentia, the latter was dissected out and its content gently squeezed directly into 100 µl of M2 medium (Sigma-Aldrich, Germany) containing 2% albumin bovine fraction V, placed in Petri dishes, and allowed to disperse at room temperature for 2 minutes (Styrna and krZanoWSka, 1995; krucZek and Styrna, 2009).

Epididymal sperm concentration
A 1:20 dilution of epididymal sperm suspension with M2 medium (Sigma) was prepared, and the number of spermatozoa in 100 squares of a hemocytometer was counted under a light microscope at 400x magnification.The average of two epididymal sperm counts was used to estimate sperm concentration.Epididymal sperm functional activity was assessed by spermatozoa motility, sperm tail membrane integrity (hypoosmotic swelling test), viability, maturity (spermatozoa without cytoplasmic droplet) and sperm head morphology.

Epididymal sperm motility
Spermatozoa motility was assessed in a hemocytometer.A small drop of sperm suspension was transferred to a hemocytometer, covered with coverslip and spermatozoa showing progressive movements were inspected.The number of spermatozoa without a cytoplasmic droplet among 200 counted spermatozoa with progressive movements from each male was reported (Seed et al., 1996).

Epididymal sperm tail membrane integritywater test
The integrity of the epididymal sperm tail membrane was determined by the hypo-osmotic swelling test (WalcZak et al. 1994;Styrna & krZanoWSka 1995;krucZek & Styrna 2009).20 µl of epididymal sperm suspension (as described in Epididymal sperm concentration) was mixed with 120 µl distilled water on a clean glass slide, then the mixture was gently covered with a coverslip and incubated for 5 min at 37° C before it was examined.The percentage of spermatozoa showing swelling among 200 counted spermatozoa from each male was estimated.

Epididymal sperm viability -eosin-Y test
The test reflects the structural and morpholo-gical integrity of the epididymal sperm membrane in human and mouse sperm (WalcZak et al., 1994;Styrna & krZanoWSka, 1995).To assess epididymal sperm viability, 20 µl of epididymal sperm suspension (as described in Epididymal sperm concentration) was mixed with 20 µl of 0.2% eosin Y, incubated for 10 min at 37° C and smeared on a slide.The percentage of spermatozoa with unstained sperm heads (viable spermatozoa) among 200 counted spermatozoa from each male was calculated.
Epididymal spermatozoa without a cytoplasmic droplet 20 µl of sperm suspension (as described in Epididymal sperm concentration) was transferred to a slide and gently covered with a coverslip.According to the classification described for mice (Styrna et al., 2002), three sperm categories can be distinguished: (1) without a droplet, (2) with a droplet on the end of the middle piece (distal droplet), and (3) with a droplet situated more proximally (proximal droplet) (Fig. 1).For the present experiments, the percentage of spermatozoa without a cytoplasmic droplet among 200 counted spermatozoa with progressive movements was given for each male.Reproductive capacity of male bank voles (Myodes glareolus Schreber, 1780)

Epididymal sperm morphology
For morphological examination a small drop of the epididymal sperm suspension was smeared on a slide, air-dried, fixed in acetic alcohol (absolute alcohol, glacial acetic acid, 3:1), and stained with Papanicolau to assess the proportions of different epididymal sperm head abnormalities.Based on our earlier study (krucZek & Styrna, 2009) for assessing bank vole epididymal sperm morphology, the following misshapen forms are distinguished : type 1-acrosomal abnormalities; type 2 -abnormalities in the distal part of the head; type 3 -serious abnormalities in the proximal part of sperm heads and type 4 -elliptic head.For statistical treatment, only classes of abnormal spermatozoa with a frequency of more than 0.5% of the total sperm in all tested animals were considered.They formed two classes: class 1 -slightly deformed with a small acrosomal part (type 1); and class 2 -other abnormalities, a group being composed of the remaining sperm defects described above, namely misshapen forms of types 2, 3 and 4.
The experimental procedures for this study were approved by the Regional Committee on Animal Experimentation in Kraków (Protocol No. 26/2007 and82/2008) acting in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and conformed to the "Guidelines for the use of animals in research" (Animal Behaviour 1991, 41:183-186)

Statistical analysis
The body weight, testes and accessory sex gland weights and epididymal sperm evaluation variables were compared by a one-way analysis of variance (ANOVA), followed by post hoc pairwise comparisons using Tuckey's tests where the ANOVA gave significant results, as data had normal distributions.For statistical treatment (1) percentages were arcsine transformed (arcsine of the square root of the proportion) (2) the following classes of epididymal sperm morphology were taken as variables: class 1; the sum of classes 2, 3, and 4; and total percentage of abnormal shapes.All results were expressed as means ± S.E.M., and p < 0.05 was considered as statistically significant.All procedures were carried out using the Statistica PL ver.8.0 statistical package.

Body and organ weights
Body weights of males increased with age up to 6 months and more.The 6M, 9M, 12M and 15M males were significantly heavier than 1.5M, 3M and 4M bank voles.Additionally, 3M, 4M and 5M males had significantly bigger body masses than did 1.5M males (Table 1).The relative weights of the testes of 1.5M males were significantly lower compared to other age groups.The biggest value of relative weight of accessory sex organs was noted for 3M males although significant differences were only observed between 3M males and the youngest 1.5M males as well as between 3M and the oldest 12M and 15M males (Table 1).

Epididymal sperm evaluation
Epididymal sperm activity was influenced by male age.Mean sperm concentrations were significantly higher in 3M, 4M and 5M males than in 1.5M, 6M, 9M, 12M and 15M males (Fig. 2).The sperm of 3M and 4M males were characterized by a large proportion of progressive motile spermatozoa and this proportion was significantly larger than in 1.5M, 5M, 6M, 9M, 12M and 15M males (Fig. 3a).Swollen epididymal spermatozoa were significantly less frequent in groups 3M and 4M than in other groups.Moreover, the 5M and 6M bank voles had significantly lower proportions of swollen spermatozoa than did 9M, 12M and 15M males (Fig. 3b).
On the other hand higher proportions of viable epididymal spermatozoa were observed in the Reproductive capacity of male bank voles (Myodes glareolus Schreber, 1780)
Sperm without a cytoplasmic droplet was significantly more frequent in the vasa deferentia of 3M and 4M males than in all other ages, and 15M and 3M males had significantly more mature spermatozoa than did 4M bank voles (Fig. 4b).Additionally, 5M and 6M males had a significantly higher proportion of mature spermatozoa than did 9M, 12M and 15M males; and 1.5M males had significantly more mature spermatozoa than did 12M and 15M males.Finally 9M males had significantly more spermatozoa without a cytoplasmic droplet than did 15M bank voles (Fig. 4b).
There were also significant differences in sperm morphology between age groups of males (Table 1).The mean total proportion of abnormal sperm was significantly lower in 3M, 4M, 5M, and 6M males than in 1.5M, 12M and 15M males.The sperm of 9M animals was characterized by a significantly lower proportion of total abnormal Means marked by the same letters differ significantly at A-O -p < 0.01, a, b -p < 0.05.sperm in comparison with 1.5M and 15M males, and 12M animals had significantly less abnormal sperm than did 1.5M males.Similarly, 3M, 4M, 5M, and 6M males had significantly lower proportions of class 1 abnormalities than did 1.5M, 12M and 15M animals; additionally, the proportion of class 1 abnormalities in 4M bank voles was significantly lower than noted for 9M males.At 9M and 12M there were lower proportions of class 1 abnormalities than at 1.5M and 15M .The sperm of 3M, 4M, 5M, 6M, and 9M males was characterized by significantly lower values of class 2 spermatozoa abnormalities than that of 1.5M males (Table 1).

DISCUSSION
A positive relationship between ageing and increase in body weight has also been documented for Spraque-Dawley rats (see for review Wang et al., 1993) while a negative correlation between body mass and age has been shown for Fisher 344 rats (baSkin et al., 1979).The high positive dependence between bank vole male body mass and testes weight observed in our experiments are in agreement with earlier observations on bank voles (ylönen et al., 2004).Since the development and secretory activity of the male accessory sex organs are under the direct control of androgens produced by the testes (franca et al., 2006), the growth of these organs in 3-, 4-and 5-months-old bank vole males could be evoked by an increase of testosterone.It has been documented for Brown-Norway rats that ageing in the reproductive system is manifested by lower serum testosterone levels (Wang et al., 1993;hardy & Schlegel, 2004).A positive role of accessory sex organs on sperm functional activity has been observed in hamsters where secretions protect sperm against oxidative damage influencing sperm viability or motility (chen et al., 2002).So for adult bank vole males, it seems to be beneficial to have larger seminal vesicles and coagulating glands.In the present study we also showed that adult bank vole males (3-, 4-and 5-month-old) produced more spermatozoa in comparison to prepubertal and aged males.Considering all parameters assessing epididymal sperm functional activity, i. e. its motility (Fig. 3a) , viability (Fig. 4a), and maturity (Fig. 4b), mature males produced sperm of better functional activity.Their sperm Reproductive capacity of male bank voles (Myodes glareolus Schreber, 1780) has been also noted in Brown-Norway rats with complete cessation of spermatogenesis in very old males (Wang et al., 1993).The opposite pattern was observed in Spraque-Dowley rats, which did not show progressive decrease in sperm concentration with age (JohnSon & neaveS, 1983).Bank voles in captivity live till about 18 months and reproduce till 15 months although their reproductive activity significantly decreased with age (from 6 month) (breeding observation M. Kruczek).In our experiment older bank vole males (to 15 month) do not cease spermatogenesis totally but produce sperm of lower quality.
Progressive movement generated by flagella is necessary for the sperm to reach the ovum in the oviduct and to initiate fertilization (mortimer, 1997).In our experiments, the swelling of bank vole spermatozoa, in response to hypo-osmotic solution, was lowest in sperm of adult, 3-and 4-month-old.A strong negative relationship between a high score in the hypo-osmotic test and pregnancy rates has been observed in men (tartagni et al., 2004).On the other hand there was no such correlation in mice (Styrna & krZanoWSka, 1995).As bank voles are more closely related to mice it can be expected that spermatozoa swelling will not have strong influence on pregnancy rate.This aspect of bank vole reproduction has not been investigated before and deserves further study using eg.Sperm Select Penetration Test (Styrna & krZanoWSka 1995).
For several mammalian species correlation between a high proportion of spermatozoa with attached droplets and infertility has been documented (gatti et al. 2004;cooPer, 2005).Fertilizing potential of spermatozoa depends on their ability to undergo capacitation and acrosome reaction and only viable spermatozoa with intact cell membrane are able to achieve this (yanagimachi, 1981).
As described in Materials and Methods, four categories of misshapen form can be distinguished in bank vole sperm.The most common abnormality is class 1.In this class, acrosomal parts of the head are shorter with very weak staining, which suggests the absence of enzymatic content, which in turn may lead to disorders in acrosomal function and consequent lower male fertility as has been shown in mice (Styrna et al., 1991(Styrna et al., , 2002) ) Spermatozoa with abnormalities in the distal part of the head, those with serious damage in the proximal part of the head, and spermatozoa with elliptical heads were rarely observed in the bank vole males from each tested age group.Since they did not exceed 0.5% of the total spermatozoa they were pooled together in one class of "other abnormalities".Our results showed that ageing in bank voles produces an increase in the frequency of altered sperm with acrosomal damage as well as in the number of other abnormal forms of spermatozoa.In hamsters, the number of abnormal spermatozoa dramatically increased in advanced-age males from 14-19% in adult to 39-50% in old males, but this increase was due more to an increase in the frequency of altered sperm than through an increase of the other misshapen forms (calvo et al., 1997).How the misshapen head morphology influences the fertility rate is still under discussion but it is generally accepted that specific head abnormalities correlate with male infertility (for reviews see WeiSSenberg et al., 1987;PeSch & bergmann, 2006).