Factors determining semen sample collection and semen quality parameters in African penguins Spheniscus demersus | Scientific Reports

Scientific Reports volume 14, Article number: 24261 (2024) Cite this article

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Our research focuses on semen collection from 42 African penguin males, grouped by age, habituation levels, and reproductive season timing. We assess behavioral and physiological responses to dorso-abdominal massage, evaluating species-specific seminal traits using conventional and advanced methods. Positive behavioral responses corresponded with successful semen collection. Young and human-reared males exhibited more favorable behaviors, and samples containing spermatozoa were more likely collected during the reproductive season. Age did not influence sample collectability or spermatozoa presence, though mature males produced higher semen volumes. Young males exhibited more sperm morphological abnormalities, especially outside the breeding season. Sperm concentration and number per ejaculate showed no significant differences across age or seasonal groups. Young males had higher motile and progressive sperm percentages, while mature males had more static sperm. Additionally, percentages of live sperm and intact acrosomes were higher in mature males. Young males and samples from non-reproductive season presented more dead sperm with damaged acrosomes. Plasma membrane integrity positively correlated with age for live sperm and negatively for dying sperm. This research demonstrates the feasibility of semen collection from African penguins regardless of habituation level, fills the knowledge gap by describing sperm morphological abnormalities, and provides insights into using flow cytometry in Sphenisciformes.

The African penguin (Spheniscus demersus), the only penguin native to Africa1, faces a high risk of extinction due to historical guano harvesting and egg collection2, along with current threats such as overfishing, oil spills, and maritime traffic disturbances2,3,4. Its population has dropped from over 3 million in the early 20th century to less than 2% of the original number2,5,6. Classified as Endangered by the International Union for Conservation of Nature since 20107, restoration efforts could benefit from the stable European ex-situ population8.

While most studies on wild African penguin reproduction focus on breeding success9,10,11,12,13,14,15, laboratory analysis of semen quality is crucial for effective population management and conservation strategies. This makes captive zoo populations valuable for research16,17,18,19. Assisted reproductive technologies (ARTs), such as artificial insemination (AI) and semen cryopreservation, are vital for preserving genetic diversity in endangered species, underscoring the importance of semen collection and quality evaluation20,21,22,23,24,25. The main methods for collecting semen from birds include dorso-abdominal massage, the cooperative method, and electrostimulation26,27,28.

Limited research exists on semen collection from captive Sphenisciformes, with only 5 out of 18 species studied16,17,18,19,29,30,31,32,33,34. The cooperative method, requiring habituation to human contact26, has been used in Magellanic31, King32, African and Rockhopper penguins18. In this technique, birds voluntarily provide semen samples in the presence of familiar humans without the need for restraint. Conversely, the modified massage technique, applied in Gentoo, African16,17, and Rockhopper penguins29,30, relies on the male’s physiological response to physical stimulation during massage and requires the immobilization of the birds. To our knowledge, electrostimulation has not been applied in penguins. Additionally, we found no studies on wild penguins’ semen collection; all studied individuals were captive-kept16,17,18,19,29,30,31,32,33,34.

Most penguin species display breeding seasonality, lasting from 4 to 15 months1. Wild African penguins breed year-round, influenced by local food availability35,36. The European population usually breeds from August to May, with egg production peaking in August-September and a secondary peak in December, though breeding behaviors can persist year-round with varying intensity35.

Traditional semen assessment techniques, such as stained smears, remain in use18,29,30,31,32,33,34; however, as advanced evaluation methods become more available, the necessary equipment is also becoming more widespread in laboratories. The Computer-Assisted Sperm Analyzer (CASA) is increasingly used to assess semen quality in various avian species37. This technique ensures greater objectivity and has shown to correlate more closely with ART outcomes than traditional methods38,39.

Previous studies on penguin species employed CASA systems to analyze sperm motility parameters. Mafunda et al.18 identified variations in sperm kinetics between Rockhopper and African penguins. In Magellanic penguins, samples from different males used for AI were analyzed33. Differences between fresh and frozen-thawed semen were documented in King32, Magellanic34, and Gentoo and African penguins16.

In Sphenisciformes, to our knowledge, only fluorochromes such as propidium iodide (PI) and SYBR-14 were utilized to evaluate plasma membrane integrity in African and Gentoo penguins via epifluorescence microscopy16,17. Similarly, these sperm characteristics were examined in King penguins using flow cytometry, but only employing PI as a fluorochrome32.

Given the limited research on semen sampling from African penguins, particularly in the wild, our objective was to investigate the feasibility of utilizing a modified massage technique to collect semen samples, considering varying levels of habituation to humans. We intended to assess physiological and behavioral responses to the massage procedure among individuals with different rearing backgrounds. This comparison aimed to assess the potential applicability of this method for wild African penguins.

Moreover, our research aimed to comprehensively assess African penguin semen characteristics using both traditional and advanced methods. To the best of our knowledge, morphological abnormalities in African penguin sperm had not been previously described; thus, our study sought to bridge this gap by employing the eosin-nigrosin staining method. CASA was employed to evaluate sperm concentration, total ejaculate sperm count, motile and progressively motile spermatozoa percentages, and kinetic parameters. Additionally, given the limited research on Sphenisciformes, flow cytometry was used to assess plasma membrane integrity, apoptosis, mitochondrial activity, DNA fragmentation, and acrosome status in African penguin sperm.

Considering access to males across various age classes and observations that African penguins at Zoo Wroclaw breed throughout the year, our study aimed to assess how age and the seasonality affect semen sample collection feasibility and seminal traits assessed through various methods.

A total of 532 semen sampling attempts were conducted, with males’ ages ranging from 1.27 years old to 21.13 years old. Of these attempts, 192 (36%) resulted in obtaining a sample, while 340 (64%) did not. The total number of procedures per male ranged from 1 to 41, with successful collections ranging from 1 to 22 and failed attempts ranging from 1 to 37 per male. Sampling was performed 248 times on keeper-reared birds and 284 times on birds reared by parents. Collection attempts were made 335 times during the breeding season and 197 times outside of it. Notably, 65 samples (76.5%) containing spermatozoa were collected during the breeding season, whereas 20 samples (24.5%) were collected outside of it. Semen samples were successfully collected from males as young as 1.31 years old, while samples containing spermatozoa were obtained from males as young as 1.65 years old. The oldest male to provide a sample with spermatozoa was 21.11 years old.

The correspondence analysis (Fig. 1) for the relationship between physiological and behavioral reactions to the massage procedure revealed that both positive behavioral reactions (“cooperative” and “calm”) exhibit a stronger association with physiological reactions described as “proper with no sample” and “sample with spermatozoa”. Conversely, “none” and “minimal” physiological reactions were more closely associated with the “uncooperative” (negative) behavior. “Agitated” behavior reveled the closest association with “sample without spermatozoa” reaction to the semen collection procedure.

Bi-plot of correspondence analysis of the physiological reaction (blue) to semen collection depending on the behavioural reaction (red) during semen collection procedure.

The χ2 test indicated no significant differences (p > 0.05) across all groups (age category, rearing method and season) regarding whether the sample was obtained or not (Supplementary Table S1). The assessment of differences across the same groups regarding the presence of spermatozoa in a sample (Table 1) showed significant disparities (p < 0.001) only between samples obtained during and outside the reproductive season, with the odds ratio being twice as high to obtain a sample containing spermatozoa within the reproductive season compared to outside of the season. Comparing behavioral reactions in age and rearing method groups, both showed significant and similar differences (p < 0.001) (Table 1). The odds ratio indicated a 60% reduced probability for positive behavior to manifest in mature males compared to young males, and in males reared by parents compared to keeper-reared males. There were no significant disparities in behavior when assessing the time of reproduction season.

Logistic regression analyses for the age of males revealed no significant differences (p > 0.05) in behaviour, sample collectability, or the presence of spermatozoa in a sample (Supplementary Table S2).

A total of 192 ejaculate samples were collected, from which 85 contained spermatozoa. Basic semen characteristics and sperm morphological parameters are presented in Table 2 as mean ± SD, along with the respective ranges.

The volumes of semen samples obtained from mature males were significantly higher than those from young males (p < 0.05). The presence of abnormal heads and spermatozoa with bent flagellum were both significantly higher in younger males than in mature males (p < 0.05). The sperm with bent midpiece were significantly more often found in samples collected in non-reproductive season (p < 0.05). None of the other sperm characteristics showed significant differences within age and season groups.

Figure 2 illustrates the morphological forms of African penguin spermatozoa. A total of 12,800 cells were evaluated to categorize these forms. Normal spermatozoa, characterized by a filiform-shaped head approximately 12 μm in length, accounted for 39.82% of the viable sperm population. Abnormal head malformations, including misshaped or abnormally wide heads (at least 1.5 times the width of a normal head), were the most common abnormality, observed in 40.57% of viable spermatozoa. The second most frequent abnormality was acrosome defects (7.15%), which included bent or detached acrosomes as well as visible acrosomal reactions. Macrocephalic heads, defined as heads at least 1.5 times longer than normal, were present in 6.67% of the spermatozoa. Other abnormalities observed included spermatozoa with a bent head, bent midpiece, or bent flagellum. Additionally, spermatozoa with coiled flagella were identified. Immature cells, characterized by round or short spermatids, were also noted.

African penguin spermatozoa morphological forms: (a) live, normal spermatozoa; (b) dead spermatozoa; (c) spermatozoa with a variety of head abnormalities; (d) spermatozoon with a bent head; (e) spermatozoon with a bent midpiece; (f) spermatozoon with a bent flagellum; (g) spermatozoon with a coiled flagellum; (h) macrocephalic spermatozoon; (i) spermatozoon with an acrosome defect; (j) immature cell (spermatid).

Sperm concentration and total sperm count per ejaculate showed considerable variation, with values ranging from 3.44 to 6163.14 × 10⁶/ml for concentration and 0.03 to 30.82 × 10⁶ for total sperm count. However, no statistically significant differences in these parameters were observed between different age or season groups. The young subgroup exhibited significantly higher values in motile, progressive, and slow sperm compared to the mature subgroup (p < 0.05). In contrast, the static sperm population was significantly greater in mature males than in young ones (p < 0.05). Additionally, the percentage of straightness (STR) of sperm paths was significantly higher in younger males than in mature ones. No other parameters showed significant differences between the age groups or across different seasons. All sperm kinetic parameters are presented in Table 3 as mean ± SD.

The sperm characteristics assessed via flow cytometry are presented in Table 4 as mean ± SD. The percentage of live sperm assessed by SYBR-14 was significantly higher (p < 0.05) in mature males compared to young ones. The percentage populations of dead and dying spermatozoa were both significantly higher (p < 0.05) in young males. There were no significant differences within the seasonal groups for this parameter. The percentage of live spermatozoa with intact acrosome was significantly higher in mature males (p < 0.001) and in samples collected during the reproductive season (p < 0.05). The percentage of dead spermatozoa with damaged acrosome was significantly higher in young males and samples obtained outside the breeding season (both p < 0.05). The percentage of dead spermatozoa with intact acrosome was significantly higher in young males (p < 0.05). None of the other parameters yielded any significant differences in any of the groups analyzed.

Among all seminal traits, only the assessment of plasma membrane integrity by SYBR-14 revealed significant correlations with age. The percentage of dying sperm revealed a negative correlation with age, while the percentage of live sperm showed a positive correlation with age (Table 5). No significant correlations were found between age and any other seminal traits assessed in this study.

Research on semen collection and quality evaluation is essential in population management and conservation strategies, as it establishes the foundation for implementing ARTs to preserve the genetic diversity of endangered species. For African penguins, this is particularly crucial as their wild population numbers are declining, with a risk of extinction in the coming decades3. While the captive African penguin population remains stable and could serve as a reservoir for future species restoration programs35, it is imperative to consider collecting samples from wild individuals as well. This approach is vital for maintaining genetic diversity, especially since not all captive individuals have fully known pedigrees and instances of hybridization have been observed8,36,40.

Semen collection from penguin species has not yet been extensively researched. In African penguins, Santiago-Moreno et al.16 and Marti-Colombas et al.17 both collected samples from 13 individuals, presumably of reproductive age, as that was not specified. Mafunda et al.18 described sampling two males, both 4 years old. All those studies are focused solely on the time of the reproductive season. Given that African penguins have an average lifespan of 25 years2,35 and typically begin breeding at around 4 years of age in the wild19,41, with potential to start younger in captivity40, our study assessed the feasibility of semen collection across a wide age range. We found that semen samples could be obtained from birds as young as 1.65 years old, continuing throughout their lives. This bears significant implications for conservation, particularly concerning wild populations where individual ages may be unknown. Our research suggests that semen samples can be collected from any individuals exhibiting signs of breeding behaviour, such as courtship displays, pair forming, or nest building35.

Other than age, the timing of the breeding season significantly affects semen sample accessibility. While most bird species, including penguins, breed seasonally1, African penguins show considerable variation in breeding seasons depending on their wild locations2,35. Additionally, captive populations breed year-round35. Our study aimed to determine the feasibility of semen collection throughout the year. We found that obtaining a sample containing sperm is more likely during the breeding season. This holds true for wild African penguins, which engage in reproductive activities and maintain pair bonds only upon reuniting at breeding grounds and cease these behaviors after reproduction when they return to the ocean2. However, our research demonstrates that in a captive colony, individuals can choose to breed and, consequently, provide semen samples even outside the presumed breeding season. This information is crucial for managing ex situ populations and using ARTs.

To our knowledge, no studies have sampled semen from wild penguins, highlighting the importance of captive populations in this research. The Zoo Wroclaw colony offers a more naturalistic setting, resembling the size and composition of wild colonies40, allowing individuals to maintain a higher degree of “wildness,” especially those raised solely by their parents. Therefore, our aim was to utilize this colony to assess this factor’s impact on the feasibility of semen sampling, providing preliminary studies for future collections from wild individuals. To our knowledge, no studies have examined the effect of habituation to humans on semen collection from penguins. In African penguins, Santiago-Moreno et al.16 and Marti-Colombas et al.17 describe the modified massage technique of Burrows and Quinn42 but do not mention the birds’ habituation levels. In contrast, Mafunda et al.18 describes a cooperative semen collection method involving two males habituated for the procedure from an early age. These unpaired birds willingly followed keepers to a secluded area, where they displayed breeding behaviour by mounting the keepers’ legs and deposited ejaculates into a petri dish. Our research confirms the effectiveness of the massage method in obtaining semen samples from both habituated and non-habituated individuals, with consistent outcomes. However, we found that the level of habituation to humans influences behavioral reactions during semen sampling, with less habituated “wild” birds showing more negative reactions. Importantly, no disruptions to normal feeding, nesting behavior, or pair bonding were observed, suggesting that this method could be safely applied to collect semen from wild populations.

In our study, ejaculate volumes from African penguins varied widely, ranging from 1 to 50 µl with a mean of 11.33 µl, which is less than half of those previously reported for this species: 21.7 µl16, 51.1 µl17, and 50 µl18. Other penguin species also show higher volumes: Magellanic penguins averaged 35 µl31, while Gentoo penguins had volumes of 36.8 µl16 and 41.0 µl17. Rockhopper and King penguins recorded the highest volumes at 240 µl29,30 and 360 µl32, respectively. Notably, these studies involved semen collection from limited numbers of individuals: 131, 218,32, 616,17,29, 1316,17, and 1430. Our study included samples from 42 males of varying ages, collected both during and outside the breeding season, which may explain the overall lower average volume observed. Additionally, birds in our colony copulated throughout the day, similar to their wild counterparts. Collecting semen samples from wild birds often yields small volumes26, and repeated copulations potentially decrease sample volumes, as documented in cranes and zebra finches43. Furthermore, our findings indicate that young males provided significantly smaller ejaculates compared to mature individuals, aligning with findings in other bird species24,43,44,45,46,47. To our knowledge, this is the first study to report age-related differences in ejaculate volumes in Sphenisciformes. In most nondomestic birds, the breeding season is brief, and ejaculate volumes are typically low at the beginning and end of this period43. In our study, ejaculate volumes from African penguins varied during and outside the reproductive season, with no significant differences observed between these periods. This suggests that the time of the year does not influence ejaculate volumes, supporting the observation that African penguins can breed at various times of the year, not restricted to the presumed reproductive season, especially when food is plentiful1,35,40.

Our study is the first, to our knowledge, to report on the viability and abnormalities in African penguin sperm morphology using eosin-nigrosin staining. We found that 69% of sperm had intact plasma membranes, indicating viability, while 31% were non-viable. This viability rate is lower compared to Rockhopper (82.9%29, 88.9%30), Magellanic (82–83%31, and 91%33), and King penguins (71.3 − 91.3%32) assessed using the same method. Interestingly, our findings align closely with viability percentages reported for African penguins using fluorescent markers, which ranged from 68.2%16 to 70.79%17. We found no significant differences in sperm viability between young and mature males, nor between samples obtained during or outside the breeding season. Morphologically normal spermatozoa averaged 39.82%, nearly half of what is reported for other penguin species: 87.5%29 and 72.13%30 for Rockhopper, 76.6%31 and 74.8%33 for Magellanic, and 78.1% for King penguins32. The low percentage of normal spermatozoa in our study is primarily attributed to a high percentage (40.57%) of abnormal heads, which were significantly more frequent in samples from young African penguins. Abnormal head formation, a primary defect occurring during sperm production30,48, may explain the high percentage observed in our study. Approximately half of the males were young, possibly with incomplete spermatogenesis, leading to abnormal heads and lowering the overall mean of normal spermatozoa. In other studied penguin species, head abnormalities are also most common29,30,31,32 but at much lower percentages (from 3.1% in Magellanic31 to 13.74% in Rockhopper penguins30). However, these studies evaluated samples solely from mature individuals29,30,31,32. Other morphological abnormalities that showed significant results within our tested groups were bent midpieces and bent flagella. Bent midpieces were more common in samples from the breeding season, while bent flagella were more prevalent in young males. These secondary defects, occurring during sperm passage49,50 or sample handling29,30,51, represented only 2.11% and 0.46% of all abnormalities, respectively, and thus are not considered crucial. None of the other abnormalities showed significant differences across our test groups. Notably, the second most common abnormality was the acrosome defect (7.15%). Including this category likely lowered the percentage of normal spermatozoa in our study compared to other penguin studies that did not assess it29,30,31,32.

Sperm concentrations varied considerably across age and seasonal groups in our study, contributing to the overall low mean value of 748.18 × 106/ml. This is lower than previously reported values for the species: 1649.3 × 106/ml by Santiago-Moreno et al.16, 1205.6 × 106/ml by Marti-Colombas et al.17, and 3274.69 × 106/ml by Mafunda et al.18. Our mean sperm concentration was similar to that reported for Magellanic penguins: 724 × 106/ml33. The notably high concentration in Mafunda’s study18 likely results from the different collection method involving un-paired males, which yielded high-quality samples and contributed to the higher sperm concentration. Santiago-Moreno et al.16 and Marti-Colombas et al.17 reported values from males of presumed reproductive age during the breeding season, explaining their higher concentrations. In our study, concentrations were higher during the breeding season but not significantly so. The mean concentration was similar for both young and mature males, indicating neither age nor time of the year influences this parameter.

Our assessment revealed that mean total motility (43.67%) and mean progressive motility (16.92%) were lower compared to Mafunda et al.18, who reported 72.06% and 43.01%, respectively. Santiago-Moreno et al.16 reported higher total motility (57.74%) but lower progressive motility (9.24%), while Marti-Colombas et al.17 presented values similar to ours for total motility (44.6%) but lower for progressive motility (7.7%). These parameters did not differ significantly between seasonal groups in our study. Interestingly, young males exhibited significantly higher percentages of total motility, progressive motility, and slow sperm compared to mature males. Conversely, mature males showed a higher percentage of static sperm. These findings suggest that young African penguins have better sperm motility parameters than mature individuals, which contrasts with findings in chickens52 where motility typically increases with age. The sperm kinetic parameters (VCL, VSL, VAP, LIN, STR, ALH, BCF) in our study were higher than those reported by Santiago-Moreno et al.16 and Marti-Colombas et al.17, except for WOB, which was slightly lower. Mafunda et al.18 showed values similar to ours for these parameters. Differences in results from Santiago-Moreno et al. and Marti-Colombas et al. may arise from their assessment of pooled samples, unlike our and Mafunda’s studies where samples were assessed individually. The mean BCF (36.16 Hz) in our study was notably higher than previously reported for African penguins (5.3 Hz16,17, 16.94 Hz18), but closer to King penguins (26.4 Hz)32, known for larger sperm head size (13.8 μm51) compared to African penguins (12.32 μm18), contributing to higher motility and kinetic parameter values16,48. The observed high percentage of head abnormalities (mainly bulb heads) and macrocephalic spermatozoa in viable sperm in our study may explain the increased frequency of sperm heads crossing the average path of a moving cell. Additionally, STR of motile spermatozoa showed a significantly higher value in young males compared to mature ones. However, these differences remain unclear due to the lack of comparative data in penguins or other bird species. Furthermore, no significant differences were found within age or seasonal groups for other kinetic parameters. These results and comparisons underscore the need for further research to establish species-specific motility and kinetic parameters and the factors that may influence them in African penguins.

Although flow cytometry is now commonly used in andrological laboratories, it is primarily employed for assessing sperm characteristics in domestic and farm animals, and in birds, mainly in poultry53,54,55,56,57,58,59,60,61,62. Few studies have applied flow cytometry to assess sperm characteristics in wild birds, including capercaillie (Tetrao urogallus)63 and Canada goose (Branta canadensis)64. Our study is the first to apply flow cytometry and fluorescent markers to assess sperm characteristics in African penguins, providing preliminary findings on plasma membrane integrity, acrosomal integrity, mitochondrial potential, apoptosis, and DNA fragmentation for the species. We found 79.92% live sperm in African penguins using SYBR-14 and PI, higher than Santiago-Moreno et al.16 (70.79%) and Martí-Colombras et al.17 (68.2%) using similar fluorochromes with an epifluorescence microscope. O’Brien and Robeck32 used PI in flow cytometry for King penguins but reported values only for frozen-thawed semen, not fresh semen. Our results were comparable to Canada goose ganders (76.3%)64 but lower than capercaillie semen (91.3%)63. We observed that mature African penguin males had significantly more live spermatozoa than young ones, consistent with viability results from eosin-nigrosin staining, though not statistically significant. Additionally, our correlation analysis indicated a positive correlation between age and sperm viability assessed with SYBR-14 and PI in flow cytometry. Our assessment using PNA revealed significantly higher values for live sperm with intact acrosomes in mature males and samples collected during the reproductive season. In contrast, morphological assessment via eosin-nigrosin staining showed a higher percentage of acrosome defects in mature males and during the reproductive season, though not statistically significant. Reports on live sperm with intact acrosomes in other wild birds vary widely, from 65.3% in Canada goose64 to 90.3% in capercaillie63, with the latter closer to our finding of 83.01%. Mitochondrial potential assessed by JC-1 in our study showed no significant differences across age and seasonal groups in African penguins. The high membrane potential of 47.19% observed in our study was notably lower than the 86.9% reported for capercaillie63. Martinez-Pastor et al.65 noted a relationship between JC-1 staining and motility, which may explain our findings given the lower sperm motility observed in African penguins (43.67%) compared to capercaillie (82.2%)66. Since correlation with motility is regulated by numerous factors66 and there are limited reports on this matter, further investigation in future studies is suggested. The sperm chromatin structure assay in our research revealed 1.83% of spermatozoa with DNA fragmentation, similar to the 1.8% reported for capercaillie63 and notably lower than the 15% in Canada goose64. As far as we know, apoptotic-like changes have not been detected by flow cytometry in any wild avian species. Our Yo-PRO‐1 assay indicated 7.76% apoptotic sperm in African penguin semen, close to the 8.6% observed in rooster sperm62. None of the last three sperm characteristics assessed (mitochondrial potential, DNA fragmentation, and apoptotic-like changes) showed significant differences between our studied groups, suggesting these parameters are not affected by male age or reproductive seasonality.

Neither age nor the level of habituation to humans influences the possibility of obtaining a semen sample from African penguins, indicating that semen samples can be successfully collected from wild individuals that display sexual behaviors. Mean ejaculate volumes are greater when collected from mature males, but the seasonality does not influence this parameter.

Eosin-nigrosin assessment indicates that African penguin semen has a lower percentage of viable spermatozoa and fewer morphologically normal sperm compared to other penguin species. Abnormal heads (more common in young males) and acrosome defects are the major abnormalities reported for this species. Sperm concentration is not influenced by either age or season. However, young African penguins exhibit better sperm motility parameters compared to mature individuals.

Flow cytometry reveals that mature penguins provide semen samples containing more viable and acrosome-intact sperm compared to young individuals, and sperm viability is positively correlated with age. This method yields higher viability results than those obtained through morphological assessment. Given the preliminary nature of these findings due to limited sample sizes, future studies should focus on expanding the evaluation of various sperm traits via flow cytometry to provide a more comprehensive understanding of African penguin semen quality.

Along with the findings from Borecki et al.40 that young age does not influence fertilization outcomes in African penguins, we can conclude that semen samples can be collected and considered for assisted reproductive technologies (ARTs) from males from an early age and throughout their lives, regardless of their level of habituation to humans. Preferably, collections should occur during the reproductive season or whenever a male displays breeding behavior. Future studies should focus on determining the optimal timing for semen collection from African penguins. Additionally, future research should determine the suitability of collected semen samples for applying ARTs such as artificial insemination (AI) or cryopreservation.

The African penguins used in this study were housed within an outdoor enclosure at the Zoo Wrocław (Wrocław, Poland), which encompasses a 900 m2 beach area and a pool with a water volume of 2,460 m3. All animals were provided with a diet consisting of frozen-thawed capelin, herring, and sprat, supplemented daily with Small Bird Supplement (Mazuri® Exotic Nutrition, PMI Nutrition International LLC, USA). All procedures conducted in this study received evaluation and approval from the internal Animal Welfare Committee (No: 3 K.2022) of the Wrocław University of Environmental and Life Sciences in Wrocław, Poland, and were conducted in accordance with the guidelines outlined by the European Association of Zoos and Aquariums (EAZA) Code of Ethics.

Semen sampling attempts were performed at least once per week for most weeks from October 2022 to January 2024. To differentiate the periods of the year, we use the terms ‘During’ (September–April) and ‘Outside’ (May–August) the reproductive season, reflecting when various individuals express breeding behaviors (nest building; courtship towards a partner, or in younger individuals, towards a potential partner). Notably, different individuals expressed breeding behavior at different times of the year. All males included in the sampling were fully fledged and had a history of breeding behavior.

A total of 42 males, including 20 young (< 4 years old) and 22 mature (> 4 years old), were assessed to determine the feasibility of obtaining a semen sample based on their age, rearing history and the time of the year.

Out of the 42 males, 21 were raised by their parents, while the other half were partially raised by keepers as part of a husbandry procedure. Birds reared solely by parents were known to remain cautious and avoid human contact during daily routines in their enclosure, which provided ample space for them to hide, maintain a safe distance, or retreat into the pool when they felt uncomfortable with human presence. During feeding, they preferred fish thrown into the pool or from a distance, rarely attempting to take fish from hand, and they did not allow themselves to be touched. These individuals were considered unhabituated (“wild”) for the purposes of this study. Males that were partially raised by keepers exhibited more courage towards handlers; they would approach them and show interest in all activities within the enclosure. During feeding, they mostly took fish from hand and allowed themselves to be touched. These birds were considered habituated to human contact.

Prior to each sampling procedure, each male was isolated from the main colony based on availability and observed breeding behavior. Males in pre-molt, post-molt, or actively molting stages, as well as those incubating eggs or raising chicks, were not sampled during this periods.

Semen collections were performed by two research personnel using Burrows and Quinn’s dorso-abdominal massage technique42 that was adapted to these species. One person, experienced in handling penguins, secured the bird, which rested with its feet stretched outward on a flat surface covered with a towel. One hand immobilized the penguin’s head, simultaneously covering its eyes and beak to prevent biting. The other hand kept one of the wings close to the penguin’s body while pressing the opposite wing to handler’s side, ensuring complete body immobilization. The second person, experienced in semen collection, stimulated the bird with simultaneous strokes on its back with one hand and abdomen with the other hand. Following the massage, gentle pressure was applied to the cloacal vent area.

Individual physiological and behavioral reactions during each sampling attempt were recorded. Male physiological reactions to the massage were documented as follows: none (no reaction), minimal (tail wiggle, cloacal inversion), proper with no sample (proper reaction: tail wiggle cloacal inversion, cloacal folds presented but no ejaculate obtained), sample without spermatozoa (proper reaction and collection of an ejaculate sample without spermatozoa), proper with spermatozoa (proper reaction and collection of an ejaculate sample containing spermatozoa). Male behavior during the massage was documented as: uncooperative (aggressive and hard to keep properly restrained during massage or at any times of procedure), agitated (does not calm down during massage and requires firm restrain throughout the procedure), calm (agitated at first but quickly calming down during massage), or cooperative (very calm at all times of the procedure).

For successful semen sampling attempts, ejaculates were collected by using a capillary extended with a tube terminated with a suction nozzle and subsequently transferred into 1.5 ml Eppendorf tubes. Semen volume was quantified through micropipetting. Small volume samples were diluted with Dulbecco’s modified medium with low glucose (DMEM) to prevent desiccation. All samples were examined within 1 h after collection. Following each collection attempt, males were returned to the main exhibit area.

Initially, all samples were evaluated under light microscopy (Nikon Eclipse E200, 200x magnification) to confirm the presence of spermatozoa. For samples with minor contamination but containing motile spermatozoa, a wash procedure was performed. This involved adding warm (37°C) DMEM medium to the specimen, allowing the mixture to settle for 5 min, during which contaminants sedimented at the bottom of the Eppendorf tube. The supernatant containing spermatozoa was then carefully transferred to a new tube. Ejaculates with high levels of contamination and immotile spermatozoa were excluded from evaluation. To minimize the influence of fecal and urate contamination on ejaculate volume and other semen parameters, samples exceeding 100 µl were not included in the assessment.

For sixty four semen sample (from 22 males; 12 mature, 10 young) containing motile spermatozoa, an eosin-nigrosin smear was prepared to assess sperm morphology. Between 2 and 3 µl of semen were placed on a slide and stained with 5 µl of eosin-nigrosin dye, left to air dry, and later evaluated under oil immersion objective (Nikon Eclipse E200, 1250x magnification) by light microscopy. To minimize morphological artifacts, such as shrinkage or distortion, associated with fixation or drying, spermatozoa were evaluated immediately after preparing the smear. The percentage of live and dead spermatozoa was determined. Cells stained pink were considered nonviable, while those without stain (white) were deemed viable. Viable sperm were further evaluated for morphological abnormalities, categorized according to descriptions from previous studies on other penguin species using the same methodology29,30,32. Abnormalities included abnormal head (swollen; abnormal shape), macrocephalic head (at least 1.5 times longer than normal), bent head, bent midpiece, bent flagellum, coiled flagellum, acrosome defect (bent or detached acrosome; acrosomal reaction), immature cells (spermatids with round heads), and other. Normal spermatozoa were classified according to Mafunda et al.18, characterized by a long, narrow, filiform-shaped head approximately 12 μm in length, a distinct but small acrosome, and a flagellum approximately 50 μm long. In each slide, 200 live cells were evaluated.

Sperm motility was assessed in 34 semen samples from 15 males (8 mature, 7 young). Total motility (MOT, %), progressive motility (PMOT, %), and sperm kinematic parameters, including curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), average path velocity (VAP, µm/s), linearity (LIN, %), straightness (STR, %), wobble (WOB, %), amplitude of lateral head displacement (ALH, µm), and beat cross frequency (BCF, Hz), were assessed using a computer-assisted sperm analysis (CASA) system CEROS II (Hamilton Thorne Biosciences, MA, USA). CASA settings for motility assessment were: progressive motility defined as > 75% STR and > 50 μm/s VAP. Spermatozoa head size detection was set within a range of 2 μm2 and 120 μm2.

Four microliters of the diluted samples were pipetted into Leja slides chambers (Leja Products B.V., Nieuw Vennep, The Netherlands) and placed on the stage warmer set at 39 °C. Five randomly selected microscopic fields were scanned at a rate of 60 frames/s to calculate the mentioned parameters. Due to the small volumes of collected samples, sperm concentration was also assessed during CASA analysis.

Flow cytometry was performed on 18 semen samples from 9 males (3 mature, 6 young) with at least 3 million total spermatozoa per sample, allowing for three primary assessments: sperm membrane integrity, acrosome integrity, and mitochondrial activity. If sperm counts were higher (5 million spermatozoa in total), additional staining protocols assessing apoptosis and chromatin integrity were conducted. The analyses were conducted using a Guava EasyCyte 5 cytometer (Merck KGaA, Darmstadt, Germany) equipped with an Argon ion 488 nm laser for excitation, and data acquisition was managed using GuavaSoft™ 3.1.1 software (Merck KGaA, Darmstadt, Germany). Sperm samples were diluted to a concentration of 5 × 106/ml, and each sample was analyzed for a total of 10,000 events. Non-sperm events were excluded based on scatter properties and were not subjected to analysis. The sheath flow rate was set at 0.59 µl/min. Fluorescent probes were excited by the 488 nm laser, with green fluorescence (dyes: SYBR-14, PNA-Alexa Fluor® 488 conjugate, monomer form of JC-1, and Yo-Pro 1) detected using an FL1 bandpass filter at 525/30 nm, orange fluorescence (J-aggregate form of JC-1) identified with an FL2 bandpass filter at 583/26 nm, and red fluorescence (propidium iodide (PI)) identified on the FL3 bandpass detector at 695/50 nm. Compensation was applied to minimize spill-over of SYBR-14 green fluorescence into the PI red channel (7.9%). Forward scatter (FSC), side scatter (SSC), and fluorescence data were collected in logarithmic mode. Spermatozoa were positively gated based on scatter properties, and all events were collected in FCS files. Dot-plot analysis was performed, and data were exported as Excel files (.xlsx) for further evaluation.

Sperm membrane integrity was evaluated using dual fluorescent probes, SYBR-14 and propidium iodide (PI), from the Live/Dead Sperm Viability Kit (InvitrogenTM, Eugene, OR, USA), following the protocol described by Partyka et al.55. Diluted samples of 300 µl were stained with 5 µl of SYBR-14 and incubated at room temperature for 10 min in the dark. Subsequently, 3 µl of PI was added 5 min before analysis. Sperm cells that exhibited green fluorescence from SYBR-14 and were negative for PI staining were considered to have intact plasma membranes (PMI), indicating viability. Cells showing red fluorescence, indicative of dead cells, were PI positive and SYBR-14 negative. Cells stained positive with both SYBR-14 and PI were considered to be in the process of dying.

Lectin PNA from Arachis hypogaea Alexa Fluor 488 conjugate (Life Technologies Ltd., Grand Island, NY, USA) was employed to assess sperm acrosome status. Samples were diluted and mixed with 10 µl of PNA and incubated for 5 min at room temperature in the dark. After incubation, the supernatant was removed by centrifugation (0.6 RCF/600 g; for 3 min), and the sperm pellets were re-suspended in 300 µl of DMEM solution. Prior to cytometric analysis, 3 µl of PI was added to the samples. Dot plots of PNA/PI stained spermatozoa displayed four cell populations: live cells with intact acrosome (PI − PNA−), live cells with ruptured acrosome (PI − PNA+), dead cells with intact acrosome (PI + PNA−), and dead cells with ruptured acrosome (PI + PNA+).

JC-1 and PI stains (Life Technologies Ltd., Grand Island, NY, USA) were used to determine sperm mitochondrial membrane potential (MMP). Each diluted sample of 300 µl was stained with 0.67 µl of JC-1 and incubated at 37 °C in the dark for 20 min before flow cytometric analysis. Prior to cytometric analysis, 3 µl of PI was added to the samples. Dead sperm cells (PI-positive) were excluded by gating on FL-3 and FL-2 dot plots. Only live sperm cells were considered for further analysis of FL-2/FL-1 dot plots. Sperm emitting orange fluorescence were classified as having high MMP, while those emitting green fluorescence were classified as having low MMP.

The Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO®-1 and PI (Life Technologies Ltd., Grand Island, NY, USA) was utilized to determine apoptotic changes in sperm cells. A volume of 1 µl of YO-PRO-1 stain was added to 300 µl of diluted samples and incubated at room temperature for 10 min in the dark. Subsequently, 3 µl of PI was added 5 min before analysis. Sperm cells exhibiting green fluorescence from YO-PRO-1 and negative staining for PI were considered apoptotic. Cells showing red fluorescence, indicative of dead cells, were PI positive and YO-PRO-1 negative. Cells stained positive with both YO-PRO-1 and PI were considered apoptotic and dead. Conversely, both YO-PRO-1 and PI negative staining indicated live and non-apoptotic spermatozoa.

The acridine orange (AO, Life Technologies Ltd., Grand Island, NY, USA) stain was used to assess sperm DNA integrity as described by Partyka et al.55, with minor changes. A diluted sample of 50 µl was subjected to brief acid denaturation by mixing with 200 𝜇l of a lysis solution (Triton X-100 0.1% (v/v), NaCl 0.15 M, HCl 0.08 M, pH 1.4), then mixed with 600 𝜇l of AO solution (6 𝜇g AO/ml in a buffer: citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl 0.15 M, pH 6), and assessed by flow cytometry. The primary population consists of spermatozoa emitting predominantly green fluorescence, indicating a normal double-stranded DNA configuration. Sperm cells positioned to the right of this main population exhibit increased red fluorescence and decreased green fluorescence compared to those in the main cluster, suggesting abnormal DNA characteristics. For each sample, calculations were conducted to determine the percentage of spermatozoa outside the main population with denatured DNA (% DFI) and the percentage of spermatozoa exhibiting abnormally high DNA stainability (% HDS). The percentage of HDS cells was determined by establishing an appropriate gate above the upper boundary of the main cluster of spermatozoa with intact DNA structure.

The relationship between physiological and behavioral reactions to the massage procedure was evaluated through correspondence analysis. The males involved in the semen collection part of our research were categorized into groups based on three factors:

Age: Young (< 4 years old) and Mature (> 4 years old).

Rearing method: Parent-reared (not habituated to humans) and Keeper-reared (habituated to humans).

Season: During (September – April) and Outside the breeding season (May – August).

For each of these groups, χ2 tests were conducted to assess differences in the frequencies of particular physiological and behavioral reactions observed during the semen sampling procedure. Physiological reactions were evaluated by analyzing whether a male provided a sample or not, and within collected samples, if they contained spermatozoa or not. Behavioral reactions were analyzed to determine if the reaction was positive (calm and cooperative) or negative (uncooperative and agitated) in each of the assessed groups.

Additionally, logistic regression analyses were conducted to evaluate the influence of age on both physiological and behavioral reactions, aiming to account for the potential confounding effect of the rearing method. The dependent variable of logistic regression model was a dichotomous variable (a male provided a sample or not, sample contained spermatozoa or not, behaviour was positive or not) and the independent variable was age expressed as a continuous variable. This procedure was necessary as the majority of males in the keeper-reared subgroup were classified as young, while most parent-reared males fell into the mature age category. This part of the analysis ensures that the significance of the age effect is verified independently of the influence of the type of rearing.

In the semen analysis part of our research, all recorded seminal traits were analyzed to identify differences between age groups (young: less than 4 years old; mature: more than 4 years old). Additionally, we compared groups based on the time of year when the ejaculate was collected, distinguishing between the reproductive season (September – April) and the non-reproductive season (May – August). The differences among groups were verified using the Wilcoxon test for independent samples. The premise for using the nonparametric method of statistical inference in this case was the lack of compliance of the distribution of the analyzed variables with the normal distribution, which in turn was verified by the Shapiro-Wilk test at the significance level of α = 0.05. Spearman rank correlation analysis among age and all recorded seminal traits was also performed. The statistical significance of all correlation coefficients was verified at the significance level of α = 0.05.

All the statistical analyses mentioned above were executed using the R67 statistical software.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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We would like to thank the Zoo Wrocław staff for all their assistance during this research. PhD student in the 6th edition of the implementation doctorate programme - Ministry of Science and Higher Education. The APC is financed by Wroclaw University of Environmental and Life Sciences. The article is part of a PhD dissertation titled “Development of cryopreservation method for African penguin (Spheniscus demersus) semen to establish a biobank for genomic resources of critically endangered species”, prepared during Doctoral School at the Wrocław University of Environmental and Life Sciences. The APC/BPC is financed by Wrocław University of Environmental and Life Sciences.

Department of Reproduction and Clinic of Farm Animals, Wroclaw University of Environmental and Life Sciences, pl. Grunwaldzki 49, Wrocław, 50-366, Poland

Paweł Borecki, Wojciech Niżański & Agnieszka Partyka

Zoo Wrocław, ul. Wróblewskiego 1-5, Wrocław, 51-618, Poland

Paweł Borecki

Department of Genetics, Wrocław University of Environmental and Life Sciences, ul. Kożuchowska 7, Wrocław, 51-631, Poland

Anna Mucha

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P.B. conceptualization, data collection and analysis, methodology, writing; A.P. supervision, funding acquisition, methodology, formal analysis, writing–review and editing; A.M. statistical analysis; W.N. supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Correspondence to Agnieszka Partyka.

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Borecki, P., Mucha, A., Niżański, W. et al. Factors determining semen sample collection and semen quality parameters in African penguins Spheniscus demersus. Sci Rep 14, 24261 (2024). https://doi.org/10.1038/s41598-024-76303-2

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Received: 23 June 2024

Accepted: 14 October 2024

Published: 16 October 2024

DOI: https://doi.org/10.1038/s41598-024-76303-2

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