The increase in wild boar numbers in recent decades is partly due to the involvement of most young females in reproduction as early as their first year of life. After the rut of adult females is over, young females are still entering oestrous as they attain maturity, prolonging the rutting period by several months. This study aimed to evaluate the effect of seasonality on the growth rate of male wild boar bodies, the growth of piglet and yearling male wild boar gonads, and sperm concentration in the epididymides. We found that yearlings' weight and body length were almost constant in summer and autumn, with a sharp increase in winter. Seasonality was also reflected in the body condition index, which rose by more than 41% between summer and winter. In terms of seasonality, the testimetric dimensions again differed significantly only in winter. Sperm were recorded in piglets weighing more than 15 kg. Regardless of the time of capture, 41% of piglets were examined as juveniles, only 6%, resp. 8% of piglets reached low or medium concentration values. While 10% of all yearling males were azoospermatic (juvenile), regardless of weight, there was evidence of seasonality in the proportion of males with measurable concentrations. These concentrations gradually increased from 62.5% in summer to 78.6% in winter. No sterile male over two years of age was noted. The results show that even in wild boars, there is a culmination not only of physical characteristics in winter but also a culmination of testimetric dimensions. Sperm already occur in 15 kg of piglets, which means they can theoretically participate in the fertilisation of female piglets.
Introduction
Wild boar (Sus scrofa) have adapted well to today's landscape thanks to their high plasticity to environmental conditions, their ability to utilise a wide range of food resources (Schley & Ropper 2003, Zeman et al. 2018, Drimaj et al. 2021a), their high reproductive potential (Frauendorf et al. 2016) and the limited effectiveness of hunting in regulating population abundance (Massei et al. 2015). In the last fifty years, European wild boars have steadily grown in number, primarily due to increasingly favourable environmental conditions (e.g. Massei et al. 2015, Vetter et al. 2020). As such, the wild boar is one of the many large European mammals able to adapt to the landscape and environmental changes and maximise its biological potential and abundance (Valente et al. 2020, Carpio et al. 2020).
This increase in population abundance has had several negative impacts, including damage to agriculture, conflicts in densely populated areas and, more recently, the spread of African swine fever (ASF) in Europe (Biondi et al. 2022) and its possible transmission to domestic pigs. Pork production is one of Europe's most important agricultural sectors and plays a vital role in the national economies of many EU countries (European Commission 2022). The recent increase in wild boar populations, therefore, pose a severe threat to agricultural and food economies at both national and continental scales (Sánchez-Cordón et al. 2018). Consequently, there has been a growing realisation that new methods need to be found to halt wild boar population growth and to eradicate wild boar from those areas most at risk from the spread of ASF. Currently, most attention is being paid to regulating wild boar numbers and addressing factors that affect wild boar reproduction and significantly contribute to population growth.
In recent years, much knowledge has been gained on wild boar lifestyle and reproductive behaviour (e.g. Vetter et al. 2016). Typically, wild boar females live with their young in family groups led by a dominant female. Under natural conditions, young males leave the family group before the birth of other piglets (Dardaillon 1988, Gethöffer et al. 2007), while their sisters stay in the group and reproduce in their second year (Brogi et al. 2021). Adult males live separately and only join the female groups for a short time during the rut, at which time oestrus is strictly synchronised (Mauget & Boissin 1987), usually lasting around one month until mid-December. In recent years, however, there has been increasing evidence that the reproductive behaviour of wild boar populations has changed, with most young females reproducing as early as their first year of life. This change appears to be a key factor contributing to the increased reproductive performance of today's wild boar populations (Ježek et al. 2011, Drimaj et al. 2020). Male wild boars are not physiologically adapted for the entry of young females into the reproductive population. However, it is clear that following the rut, young females still enter oestrous as they attain maturity, prolonging the rutting period by several months (e.g. Drimaj et al. 2020, 2021a). This situation means that males must now be physically prepared for fertilisation throughout most of the year (Delcroix et al. 1990). On the other hand, there is a large difference in body size between adult male and young female wild boars, which could hinder successful mating (Drimaj et al. 2020). Generally speaking, large males live alone and fight for groups of females, after which the large male prevents smaller males' access to females (apparently unsuccessfully, as multiple paternity has been demonstrated; Müller et al. 2018). At the same time, however, the large male is unlikely to be able to mate with the small females, who would typically remain unfertilised despite having reached sexual maturity. Nevertheless, many of these young females are successfully fertilised during the first winter of their lives, i.e. at approximately 7-9 months of age (e.g. Gethöffer et al. 2007, Servanty et al. 2009, Drimaj et al. 2020). However, there is a complete lack of data on which males fertilise these young females in late winter and spring. Most wild boar reproduction studies have focused on females, and almost nothing is known about the importance of males for population growth, despite successful conception not only being conditioned by the female in oestrus but also by males that can successfully fertilise females at a given time.
Previous studies have shown that males grow faster and enter puberty at an earlier age in an environment providing enough food, lack of disturbance and shelter (Drimaj et al. 2019, Maistrelli et al. 2021). In addition, accurate histological and morphofunctional investigations of the testis and the duration of spermatogenic events in sexually mature wild boars have also been conducted (Almeida et al. 2006, Maistrelli et al. 2021). These studies suggest that in both wild boar and their domesticated cousins, there is a clear adaptation to environmental conditions in relation to seasonal reproduction. However, while this is relatively well-researched in females (e.g. Mauget & Boissin 1987, Kozdrowski & Dubiel 2004, Malmsten et al. 2017), more detailed studies on male seasonality are needed.
This study aimed to evaluate the effect of seasonality on i) the growth rate of male wild boar bodies, ii) the growth of piglet and yearling male wild boar gonads, and iii) sperm concentration in the epididymides. The following hypotheses were defined: 1) both physical and testimetric parameters increase in males with the onset of the main reproductive period (i.e. at the end of the calendar year); 2) also here: there is a sharp increase in sperm concentration in the epididymides; 3) all yearlings and adult males have a high concentration of sperm in the epididymides.
Material and Methods
Study area
Samples were obtained from the Soutok game preserve (48.6802158 N, 16.9443242 E; 151-153 m a.s.l.), the largest game preserve in the Czech Republic, at ca. 42 km2. Situated between the Rivers Morava and Dyje, the preserve includes large areas of floodplain forest primarily made up of oak (Quercus sp.) and ash (Fraxinus sp.), along with several softwood species (e.g. willow Salix sp., poplar Populus sp.). The area is covered by semi-natural floodplain forest (80%), meadows and pastures cover 12.6%, and water bodies make up 7.2% of the area. The mean annual temperature is 9.7 °C, and the mean annual precipitation was 496.8 mm for the study area (Nezval et al. 2020). Prior to this study, ungulate density within the forest was estimated at 11.3 ind./km2 (red deer Cervus elaphus, fallow deer Dama dama, roe deer Capreolus capreolus and wild boar) with wild boar at 7.6 ind./km2 (Mikulka et al. 2018), representing one of the highest carrying capacities in Central Europe (Zeman et al. 2018). In addition to natural food, ruminants have access to small fields with winter rapeseed, wheat, barley, maize and potatoes. Ruminants are fed with artificial feed (maize, oats and barley), but their availability is minimal for wild boars, as the feed is presented in high troughs. Wild boars are shot during hunts throughout the year, regardless of age or gender (on average, 600 wild boars are harvested annually). The game preserve is permanently fenced off, but the fence is permeable to wild boar, and they migrate to the surrounding fields during the growing season.
Data collection and analysis
Samples (reproductive organs) were collected from wild boar males between 20 April 2019 and 31 January 2020. From February to March, wild boar hunting did not occur because the females are usually pregnant (this follows from the hunting regulation). At the time, we noted the date of hunt, body length (BL; the distance from snout-tail), body condition index (BCI; Drimaj et al. 2020), the height of back fat measured at the 12th rib level after incision of the skin, together with Musculus longissimus dorsi;), dressed weight (BW) and age (calculated based on tooth development and eruption (Matschke 1967); piglets: < 12 months, yearlings: 13-24 months, adults: > 24 months). The scrotum, including the testes, epididymides and 5 cm of vas deferens, were separated from the body and stored in a freezer at a constant temperature of –20 °C. After thawing, testicular biometric values were assessed for both left and right gonads, according to (Věžník et al. 2004), i.e. length of testes, including epididymis (LTIE); length of testes and epididymis head (LTEH); length of testes (LT); thickness of the testes along the craniocaudal axis (HT) and testicle width along the medio-lateral axis (WiT). After dissection, the weight of the testes (WeT), epididymides (WE) and tunica albuginea (WTA) were determined. Testicular volume was calculated as Volume = length × width2 × (π/6), according to Bekaert et al. 2012. The gonadosomatic index (GSI) was calculated as GSI = WeT/BW × 100, according to Barber & Blake 1991.
To assess whether an individual was already producing sperm and at what volume, the epididymis and part of the vas deferens was isolated and cleaned, with the epididymis flushed according to Martinez-Pastor et al. 2006. Flushing was performed with a 21G blunt needle using 2 ml of saline. After five minutes of sedimentation, a sample was taken from the sediment to quantify sperm content. Sperm concentration in the lavage sediment was determined using the Sperm Class Analyser® CASA system (Microptic SL, Barcelona, Spain) and a Nikon Eclipse E200 microscope (Nikon Instruments Inc., Melville, NY, USA). Based on the average concentration value obtained from both epididymides, wild boar males were divided into four groups, 1) azoospermatic and considerably oligospermatic lavages, 2) lavages with a concentration of 1-20 million sperm in ml sediment, 3) lavages with a concentration of 20-100 million sperm in ml sediment, and 4) lavages with a concentration of more than 100 million sperm per ml of sediment.
One-way ANOVA was used to assess seasonal dependence of individual body and testicular parameters, while relationships between biometric values were tested using Pearson's correlation coefficient. Unless otherwise specified, the significance level was set at 0.05, meaning that the null hypothesis was rejected at P < 0.05. All statistical analyses were performed in the STATISTICA 12.0 (StatSoft, Tulsa, OK, USA) statistical package.
Results
Body parameters
A total of 125 male gonads (from 125 males) were analysed, of which 62 were yearlings, 46 piglets and 17 adults. Seasonal comparisons of piglet (Table 1) weights were not possible due to the high variability resulting from year-round and unevenly distributed births. Likewise, it was not possible to assess seasonal differences in adult body weight due to the relatively low numbers. Thus, seasonal differences in body parameters could only be assessed for yearlings (n = 62). Yearling BW remained relatively constant during summer and autumn and increased significantly in winter (ca. 10 kg higher than in autumn or summer; P < 0.01). BL showed a similar trend (in relation to weight: R = 0.83, P < 0.01), with yearlings ca. 8 cm longer in winter (compared to summer and autumn). BCI showed an increasing trend as winter approached, being significantly related to both body length (R = 0.62, P < 0.1) and weight (R = 0.48, P < 0.01). The main change in BCI occurred between summer and winter when the average BCI increased by more than 41% (P < 0.01). Given the average weight of 40.34 kg, there was no difference in BCI between autumn and winter; only summer differed from this period (ANCOVA, F2,58 = 3.54, P < 0.05). There was a significant correlation between the month of the hunt and BCI (R = 0.52, P < 0.01).
Table 1.
Mean body and testicular parameters in wild boar age classes and in yearlings by season.

Table 2.
Correlation between body and mean testicular parameters in wild boar males.

Table 3.
Correlation between body and mean testicular parameters in wild boar yearling males.

Testimetry
Mean body and testimetric values were calculated for piglets, yearlings and adults, along with mean seasonal values for yearlings (Table 1). All biometric dimensions differed significantly across age classes (all P < 0.05; Table 1). Testimetric R values indicated the strongest correlations with BW, less strong with BL and least with BCI (Table 2; all P < 0.01). Mutual relationships between testimetric parameters showed strong correlation, with R values ranging from 0.67 to 0.98 (all P < 0.01). While all yearling seasonal parameters showed a slight, though non-significant, increase between summer and autumn, the same parameters had increased significantly by winter (P < 0.05; Fig. 1). As yearling growth rate is slower than that for piglets, testimetry tended to be less linked with body biometrics (particularly body length), with some values being non-significant or showing low (P < 0.05) significance (Table 3).
Spermatozoa
Regardless of the season in which the hunt took place, 41% of piglets were diagnosed as azoospermatic (juvenile). This figure rose to 81-92% with the inclusion of piglets with sporadic sperm incidence, leaving just 6 to 8% with low to medium concentrations (Fig. 2). While we noticed no significant correlation between sperm concentration in the epididymis and body weight, body length or BCI, the proportion of piglets with low to medium sperm concentration in the epididymis increased rapidly alongside body weight as the end of the calendar year approached. Sperm were recorded in 27 piglets weighing more than 15 kg. While 10% of all yearling males were azoospermatic, regardless of weight (range 19-45 kg), there was evidence of seasonality in the proportion of males with measurable sperm (n = 42), with concentrations gradually increasing from 62.5% in summer to 78.6% in winter (Fig. 3). Individuals weighing less than 45 kg tended to have lower sperm concentrations, while those heavier than 45 kg tended to have medium to high sperm concentrations (P > 0.05). While body parameters other than weight did not affect sperm concentration, there was a significant relationship between sampling time and BCI (R = 0.52, P < 0.001). Adult males had measurable sperm concentrations (i.e. azoospermatic males recorded), regardless of sampling time, with 82.3% having moderate to high sperm concentrations (Fig. 2).
Discussion
Piglets grow rapidly in their first year of life, following which there is a differentiation in energy management strategies between the sexes. Females invest a higher proportion of their energy input into reproduction, while males continue to use most of their resources for growth (Brogi et al. 2021). This period of energy differentiation usually overlaps with the spring rut, a period when piglets are becoming sexually mature and the sow excludes young males from the group to avoid mating between siblings (Keuling et al. 2016). At this time, inexperienced young males wander free in the environment without the protection of experienced mothers and frequently become easy prey for hunters. Indeed, in our study, 60% of all yearlings were shot between January and April. During this period, hunters purposely target bigger piglets, with 90% of piglets shot in the study area weighing over 15 kg. This high hunting pressure on older piglets and yearling males may explain the relatively low number of adult males shot.
Hunting, therefore, is a crucial factor affecting the distribution of wild boar in the environment (Drimaj et al. 2021b, c), and especially for the development of population dynamics through the selection of individuals that can participate in reproduction (Ježek et al. 2011, Keuling et al. 2016). “Natural” factors that could affect wild populations in the Soutok game preserve include flooding and population density. Flooding, natural or controlled by water managers, can cause significant stress to pregnant females and increase postnatal piglet mortality due to drowning or hypothermia (J. Drimaj et al., unpublished data). However, most floods in this floodplain reserve occur around March during the spring snowmelt (Klimo et al. 2013, Brázdil et al. 2014). Thus, the winter rut period is largely unaffected by flooding.
In a previous study conducted across the Czech Republic, it was shown that fertilisation of female yearlings and adult sows occurs primarily during November and December, though some females may already have been fertilised in October (Drimaj et al. 2020), this variability in the onset of oestrus being tied to changes in the daytime light phase (Smital 2009). At this time, the dominant adult males become fully involved in the reproductive process (Brogi et al. 2021). During this rutting period, the body weight of adult males tends to decrease rapidly (Mysterud et al. 2004), with individuals often losing up to 20% of their original weight (Heptner et al. 1988). This effect is likely due to the constant search for females in heat and the high energy costs of competitive interactions. Weight loss in yearlings at this time is much less pronounced, while that in piglets is almost negligible (Brogi et al. 2021). However, Brogi et al. (2021) have suggested that this weight loss may not be due to activity-related depletion during the rut but rather due to a response to long-term shortages of nutrient-rich food of natural origin.
Our results showed that yearling body size in the Soutok game preserve increased most at the onset of winter. Previous studies of wild boar food availability in the study area have shown that the main dietary components in the summer are natural resources such as grasses, roots and tree fruits. However, in the autumn, the proportion of natural food resources decreases and is replaced by artificial resources, especially maize and grain (Mikulka et al. 2018, Zeman et al. 2018, Drimaj et al. 2021a). The Soutok game preserve supports large numbers of ruminants, such as roe deer and fallow deer, whose diet is mainly supported by supplementary feed provided by the hunting organisation. While this food is not explicitly aimed at supporting wild boar, the species has been shown to feed intensively on this supplementary food (Mikulka et al. 2018, Zeman et al. 2018), meaning that wild boars usually have plentiful food, even when natural food resources are depleted (Oja et al. 2014). In mast years, acorns may also make up a significant proportion of the diet; however, owing to the high density of ruminant ungulates in the study area, most of these acorns will have been consumed by the end of November, even in mast years (Mikulka et al. 2018). From October to November, wild boars in the reserve have an energy-rich diet, which is reflected in a sharp increase in adipose tissue thickness (especially subcutaneous fat) and an increase in weight and body length (see also Brogi et al. 2021).
Surprisingly, the average winter weight of adult wild boars in the study area was somewhat lower than that recorded at other sites with less food availability, with yearlings and adult males being around 30 kg lighter than their counterparts in the Czech Republic (the average Czech yearling male weighed 79 kg and an adult male 106 kg; Drimaj et al. 2019) and other parts of Europe (Herrero & De Luco 2003, Delgado et al. 2008, Sprem et al. 2011). As body weight was highly correlated with testicular dimensions in our study, it is no surprise that testimetric values were also below the national average. However, they did not differ in proportion to body weight. BCI did not affect the testimetry.
Our results confirmed sperm occurrence in piglets whose dressed weight exceeded 15 kg (approx. 19 kg live weight). However, in our previous study examining wild boar populations across the Czech Republic, the first occurrence of sperm was generally described in piglets weighing ca. 29 kg (Drimaj et al. 2019). Likewise, a recent German study (Maistrelli et al. 2021) found that puberty occurred at around nine months (i.e. at 29 kg body weight), with the onset of sexual maturity at ten months (34 kg). As the peak in piglet births occurs in March and April, it follows that male piglets reach sexual maturity at the end of the year or the beginning of the following year. Therefore, it is highly probable that they would be capable of participating in the rut from this point on.
As such, the sows likely exclude adolescent males from family groups at this time to prevent inbreeding between related piglets/yearlings (see also Truvé & Lemel 2003).
As piglets already have sperm in their testicles by the end of the year, they are likely capable of siring offspring. These individuals may also be capable of fertilising adolescent females, whose rut follows that of the yearling females and adult sows serviced by adult boars and yearling males. As the boar must actively accompany and control females in rut in his family group over large areas, the mating period can be demanding for boars; hence, these boars may be less able to rigorously control adolescent female piglets in heat, providing an opportunity for adolescent males. Indeed, such behaviour was observed during long-term thermal imaging observations of wild boars between 2017 and 2022 (J. Drimaj, pers. observ.).
Our results indicated that sperm concentration in male yearlings increased toward the end of the year in line with the growing numbers of females entering oestrus (Brogi et al. 2021), with peak sperm concentration coinciding with the point when the maximum number of females were in heat (Schopper et al. 1984, Mauget & Boissin 1987, Marchev et al. 2003, Kozdrowski & Dubiel 2004, Sancho et al. 2004, Smital 2009). Note, however, that piglets may be born sporadically throughout the year, meaning that while sperm concentrations vary through the year (Colenbrander & Kemp (1990) estimated quantitative changes of around 25-30% over the year in adult males), some males will always have enough sperm to fertilise females regardless of photoperiod (Kunavongkrit et al. 2005). Similarly, the most significant decrease in sperm production occurs during female anoestrous, which, according to the literature (e.g. Kozdrowski & Dubiel 2004), is caused by heat stress in summer months. Under summer conditions of heat stress, egg cells in non-lactating adult females fail to mature and the follicles do not grow, while females in heat have a lower chance of successful fertilisation and pregnant females have higher prenatal mortality rates (e.g. Delcroix et al. 1990). Male sperm production also declines in the summer, and the sperm display poorer motility and a growing proportion of morphological abnormalities (Kamanová et al. 2018). After summer, it takes around two-to-three months for baseline levels to be restored.
The current population explosion in wild boar populations poses a real risk to European commercial pig farming due to the possible spread of ASF; thus, there is an increasing need to find effective means of controlling European wild boar numbers. However, this is complicated by the high reproductive capacity of wild boars of every age group and the high number of females relative to males in the adult population, making the hunting of adequate numbers of females challenging (Kamler & Drimaj 2021).
Acknowledgements
The research was supported by the Specific University Research Fund MENDELU, No. IGA-LDF-22-TP-006. Thanks to Kevin Roche, a native speaker, for the language correction.
Author Contributions
The study was conducted with contributions from all authors. J. Drimaj, J. Kamler and Z. Rečková designed and planned the study. J. Drimaj, J. Kamler, Z. Rečková and O. Mikulka conducted the field measurements and laboratory work. J. Drimaj performed the statistical analyses. J. Drimaj, J. Kamler and Z. Rečková analyzed and interpreted data. Finally, J. Drimaj prepared the draft of the manuscript.
Data Availability Statement
The datasets used and analysed during the current study are available from the corresponding author upon reasonable request.