Identification of honey bee sperm structures following the use of various staining techniques

The honey bee plays an enormous role in human life, not only as a producer of bee products but above all as an integral element of the world of plants. Most studies of bees concern the Carniolan honey bee (Apis mellifera carnica), one of the most popular species kept for honey. The Buckfast breed of honey bee, the result of crossing Apis mellifera ligustica with Apis mellifera mellifera (3), is increasingly popular among beekeepers because it possesses many advantages. There is no recognised breed standard for the Buckfast bee. Artificial insemination of bees is currently successfully used on a large scale, mainly for breeding work, but also for research purposes. The literature contains no information on the analysis of the semen of Buckfast bees and relatively few studies on the breed generally. Many studies indicate differences in semen traits depending on bee genotype (44, 51). This is also confirmed in a study on bees which showed significant differences in the number of sperm in semen obtained from drones representing various genetic lines, from 2.21 × 10⁶ to 4.11 × 10⁶ (43). While the morphology and morphometry of the sperm of mammals is well known, there has been very little research of this type on the sperm of insects, including the honey bee. For mammals, there are specific, precise spermiograms that identify various morphological sperm defects. In the case of modified sperm, which are produced by most invertebrates and have the most complex and varied structure, the determination of specific morphological defects is very difficult. Comprehensive semen analysis focusing on sperm morphology and morphometry is one of the most important tools for improving honey bee lines. Many studies indicate that male fertility is linked to sperm dimensions and shape and to DNA damage (4, 10, 37). Elimination of abnormal sperm is especially important in the case of honey bee breeding, because a single procedure of artificial insemination with semen collected from 3–6 drones causes the queen to retain from about 4.5 to 5.7 million sperm in the spermatheca and thereby provides male gametes sufficient to fertilise ova throughout her life, as sperm can be stored there for several years. Hence insemination of the queen bee with drone sperm of poorer quality may result in losses or ultimately in a weaker colony (39, 51).

In laboratory practice, various staining methods are used for rapid prediction of male fertility based on observation of the ultrastructure of sperm. Papanicolau staining is often used to evaluate human semen (34). For the evaluation of animal semen, a simple technique for staining smears with a complex of eosin and gentian dye is used (2, 24), while SpermBlue is used to analyse the semen of both humans and animals (49). Insect sperm are difficult to study, which means that the choice of staining method takes on great importance. However, it is not just the technique itself that is important, but the accuracy of the evaluation of sperm morphology, which depends on careful slide preparation and fixing and staining of sperm to minimise any effect on the morphometry of the head and the entire spermatozoon (7, 29, 32, 35). Thus the choice of staining technique is very important; it should interfere with the cells as little as possible (30) while at the same time clearly show the boundaries of the head and other elements of the sperm structure so that each of these parts can be accurately identified. Analysis of sperm morphology is a subjective assessment and is difficult to standardise. The aim of the study was to find a method for staining the semen of honey bees of a Buckfast line that would enable the identification of as many details of the sperm structure as possible without significantly altering its dimensions, which would remain within the accepted limits for drone sperm. An additional aim of the study was to perform a comparative analysis of the morphometry of sperm following the use of various techniques for staining drone semen.

The bees studied were kept in hives of the ‘Wielkopolski’ type commonly used in Poland, with dimensions of 360 × 260 mm. They are located in rural parts of eastern Poland, where there are no large monoculture plantations (e.g. rapeseed or buckwheat). Food sources available to these honey bees in early summer are black locusts, fruit trees, deciduous honeydew from various plants, and meadow plants. In summer, Polish honey bees have access to linden, borage, and other herbs. The research material consisted of the semen of drones of the Buckfast bee breed and B4 (GRP) line from a registered apiary (http://pawluk.net.pl/). At the start of the mating season (delayed by Polish weather conditions in 2022 from June 1 to August 15, 150 drones were randomly selected for the study. Semen was obtained from sexually mature drones at the age of 30 days by artificially everting the endophallus after pressing the thorax and then the abdomen according to the method described by Cobey et al. (5). Semen was collected directly into sterile Eppendorf tubes and diluted by adding 0.5 cm³ of 0.6% of NaCl solution to the semen and gently mixing. Microscope slides were prepared immediately following dilution. A drop of semen was placed on a degreased microscope slide for easier identification of sperm, and then a smear was prepared. The slide was left to air-dry and then stained. The morphology and morphometry of the sperm were assessed on slides prepared by three staining methods according to the protocols described by the instrument manufacturer, modified for bee semen (49), using the Sperm Class Analyzer system (Microptic S.L., Barcelona, Spain). SpermBlue staining (Microptic S.L.) was carried out as follows: slides with smears were placed vertically in a staining jar with SpermBlue stain for 5 min. To remove the stain, the slides were slowly immersed in distilled water and left to dry in a vertical position. Finally, the slides were sealed with a cover slip using distyrene plasticiser and xylene. Staining by the Sperm Stain method was carried out using three ready-to-use kits for the semen smear (Kit 1 – hexamethyl-p-rosaniline methanolic solution; Kit 2 – xanthene buffered solution; Kit 3 – thiazine buffered solution). A slide with a semen smear was immersed five times for 1 s in each of the stains and then left to dry. After staining, the slide was rinsed with distilled water and left to dry. For eosin-nigrosin staining, semen was added to a small Eppendorf tube with BrightVit solution (Nigrosin-Eosin), mixed thoroughly for 15 to 20 s, and left for 10–15 min at 37°C. Then a drop of the semen + BrightVit mixture was placed in the centre of a microscope slide, and a smear was prepared and left to dry. The sperm cells were evaluated with a Nikon Eclipse E200 microscope (Tokyo, Japan) and a Basler ACA 1300-200 UC camera (Ahrensburg, Germany) configured to work with Sperm Class Analyzer system version 6.5.091 (Microptic S.L., Barcelona, Spain). The microscope was equipped with a 60× bright-field objective. In order to increase the accuracy, the measurements of individual sperm parts were made manually. In each ejaculate, morphometric measurements were made of 50 randomly selected sperm. A total of 7,500 sperm were evaluated. Depending on the staining technique and the possibility of discerning individual sperm structures, the following morphometric parameters were measured: acrosome length, nucleus length, total head length, midpiece length, tail length (without midpiece), the total length of tail with midpiece, and total sperm length.

The data from the morphometric measurements of the spermatozoa were stored in a database and exported for further statistical analysis. Statistical differences between the samples were tested using Tukey’s test in STATISTICA version 12.5, (StatSoft Inc., Tulsa, OK, USA). The significance level was set at P≤0.05.

Photographs were taken of drone semen stained by various techniques, showing how the chemical substances used to prepare the slides revealed various elements of the sperm structure with differing clarity. This dissimilarity necessitates accounting for the differences in the colouring and sharpness of the image of the cells resulting from each slide preparation technique when analysing sperm morphometry. The most details of the drone sperm structure could be seen in staining with the eosin-nigrosin complex. This method made it possible to identify all structures of the sperm cell. The background of the slide was stained but fairly light, so it did not hinder the analysis. The colour of the acrosomal part, which adheres to the anterior pole of the rod-shaped nucleus, was similar to that of the background but easy to identify (Fig. 1a and b). The nucleus was much lighter so that the length of the acrosome could be precisely measured. The most interesting element observed in this staining technique was the sperm midpiece. This part of the sperm was clearly stained dark violet, and the mitochondrial derivatives formed a distinctive spiral whose diameter increased towards the flagellum. This effect was only observed in this staining technique and was seen in nearly all sperm visible in the fields of view of the microscope. In addition, staining with eosin and nigrosin revealed the uneven distribution of sperm proteins in different parts of the tail. It was not possible to identify so many details of the sperm structure with the Sperm Stain method (Fig. 2).

Fig. 1

Fig. 2

The background was light and nearly unstained and did not impede the evaluation. The acrosomal part was pale violet, but distinctly visible so that its area could be measured. Staining by the Sperm Stain technique coloured the nucleus dark violet, so that the image was sharp and clearly visible, facilitating measurement. This method identified the tail very well, giving it a violet colour, but it was not possible to discern the boundary of the midpiece. SpermBlue staining revealed the fewest details of the drone sperm structure (Fig. 3).

Fig. 3

The background of the slide was light, and the sperm were uniformly stained so that identification of individual structures was impossible. The acrosome, nucleus, midpiece and tail were pale and poorly visible. Only the total length of the sperm could be measured. In our previous research (19) using staining with eosin and gentian violet complex (Fig. 4), the sperm structures possible to identify were similar to those possible in staining with Sperm Stain.

Fig. 4

The background of the slide was much lighter, facilitating analysis, while the entire sperm was coloured violet with a distinctly darker nucleus so that its boundary could be precisely identified. Table 1 only includes measurements that were made possible by the staining of sperm structures, in order to present more reliable results.

Table 1 presents data comparing the morphometric traits of drone sperm using different techniques for staining microscope slides. The data show that the staining method and thus the chemical reagents used affect the dimensions of drone sperm. The smallest dimensions of sperm were found when staining with SpermBlue and Sperm Stain, as they had the shortest acrosome length and nucleus length. The heads of sperm stained with Sperm Stain were 0.23 μm shorter than those of sperm stained with eosin and nigrosin complex (P≤0.05). Only in the case of eosin-nigrosin staining was it possible to measure the sperm midpiece, which was on average 30.63 μm long. It is difficult to compare the results for tail length, as in the case of eosin-nigrosin staining the length of the tail did not include the midpiece. Sperm stained with Sperm Stain were also the shortest in total, being more than 10 μm shorter than sperm stained with eosin-nigrosin and approximately 6 μm shorter than that of sperm stained with SpermBlue. However, the differences were not found significant statistically.

Sperm are considered normal if they fall within the classification for a given species, which includes the shape and size of the head, midpiece and tail (20). Research indicates that the size and shape of the sperm cell significantly influence its functionality, including the acrosomal reaction (33) and binding to the zona pellucida of the oocyte (13). Honey bee sperm, like the sperm of most invertebrates, are modified sperm. The head contains a rod-shaped nucleus and an elongated acrosome located in front of the nucleus (27), and the midpiece is highly elongated. Further in-depth research on insect sperm is justified because the complexity of the structure of modified sperm is the result of a very complicated spermatogenesis process (16). It takes place inside cysts that fill the seminiferous tubules of the gonad. Spermiogenesis in insects takes place in two stages. In the first stage, the spherical, post-meiotic nucleus is transformed in the spermatids into a very elongated sperm nucleus. The transformation of the nucleus is associated with the effect of the sheath of microtubules formed around it. As a result, the nucleus is elongated and its volume decreases, while the chromatin is highly condensed (16). At this stage, the acrosome is formed, as well the axoneme and two mitochondrial derivatives that will form the flagellum. Mitochondrial derivatives are of unequal size and lie parallel to the axoneme (27). The fully formed axoneme runs along the flagellum, forming its axis. In most insects, the flagellum consists of microtubules 9 + 9 + 2 and two mitochondrial derivatives running along it (17, 51, 52). The second stage of spermiogenesis is a process of individualisation, beginning after the elongation of the spermatids is complete. It involves the breakdown of syncytial complexes of spermatids into separate cells when the cytoplasmic bridges connecting them break (16). Finally, mature honey bee sperm are threadlike cells that narrow at the ends (27, 38), as shown in Figs 1, 2, 3 and 4. The sperm of Apis mellifera, as of most insects (41), are fairly long: the total sperm length is 250–270 μm. They are 0.7 μm wide (38). According to Peng et al. (38), the head of a drone spermatozoon with a normal structure is 10 μm long and 0.4–0.5 μm wide. The total length of the acrosomal complex is 5 μm (26). These values for morphometric lengths are consistent with our results; however, the sperm stained in our experiment had somewhat shorter heads, most likely due to the composition of the chemical reagents used and the smear preparation methods. Tarliyah et al. (47) reported shorter honey bee sperm than in the present study, whereby the average sperm length was 217.57 μm, and the length of the head was 7.52 μm.

The present study showed that the staining method can significantly influence the results of morphometric measurements. Hence the lack of standardisation of staining techniques for assessment of the morphology and morphometry of the sperm of specific animal species seems to be a significant problem. The commonly used complex of eosin and gentian violet dye is acidic (19). Most proteins of the sperm head are alkaline, so following the use of various stains the acrosome is coloured differently from the post-acrosomal region. These differences can be seen in the photographs presenting the results of the present study. The use of various chemical reagents, and in many cases even the fixation of the semen, can affect the dimensions of individual sperm structures. Alcohol can dehydrate and shrink the sperm head, and incubation of slides in physiological saline solution can cause the head, midpiece and tail to swell. However, there has been research showing no negative effect on human sperm dimensions following preparation of slides using SpermBlue. In the results of that research, the morphometric measurements of the sperm were the most similar to the results obtained in fresh, unstained semen probably because of substances having an isoosmotic effect on semen (31). In the case of bee sperm, however, this technique did not produce satisfactory results, as it failed to differentiate the individual elements of the sperm cell. Differences in sperm dimensions in species besides bees, especially in the case of modified sperm, may be determined by the cytoskeleton of the sperm head, which consists of specific proteins and a nuclear envelope. Changes in the arrangement of actin fibres in the head may be caused by the means of fixing the smears and the staining method (9). A study by Collins and Donoghue (6) confirmed that bee sperm should be stained with a variety of techniques, as each of them may reveal different details of the ultrastructure of the sperm or morphological defects.

In breeding work, analysis of the morphometric measurements of sperm is a significant element of observations, as many studies indicate that sperm morphometry is associated with fertility in males of various species (1, 25, 40, 45). Gage (11) explains that the shape of the sperm head is an important factor influencing its hydrodynamics and suggests that sperm with thinner and more oval heads have better motility. It has also been suggested that not only the size of the sperm head affects fertilisation, but also the dimensions of the tail and midpiece. Sperm with a longer tail have a greater fertilisation capability due to increased motility (1). It has been concluded that longer sperm may be an adaptation increasing their competitiveness (18, 53). The competitiveness of sperm can be an evolutionary force leading to differences in their morphology and function due to strong selection pressure (53). Research on the competitiveness of sperm has also been carried out in insects (23). One of the mechanisms of sperm competition is the incapacitation of rivals: when the sperm of different males meet, substances present in the seminal fluid of one male cause the sperm of other males to die (21). It would seem that this might have the negative consequence of increased sperm mortality. However, Tofilski et al. (48) conclude that mixing the semen of different drones does not negatively affect the viability of sperm, which is confirmed by the research of other authors (46, 50). Contrasting results were obtained by den Boer et al. (8), possibly because of differences in the method of semen collection (48). However, in assessing the viability of sperm taken from a drone, we should bear in mind the valuable finding of Gençer et al. (15) that the viability of sperm collected into a needle for artificial insemination is about 7% lower than that of sperm in the seminal vesicles, due to the unavoidable reduction in their natural viability resulting from the pressure applied during eversion of the copulatory organ.

In addition, sperm are prone to having clearly visible malformations. Lodesani et al. (28) identified certain morphological abnormalities in drone sperm, such as various aberrant tail forms, including coiled, frayed, and double-ended forms, but they were not precisely described and it is difficult to specify their cause. The authors suggest that some sperm anomalies may arise during the cryopreservation of semen. Some abnormalities were observed in the present study as well (Fig. 3). They may have been generated by the slide preparation technique, as morphologically altered sperm have most often been found in smears stained by the SpermBlue technique. Our previous research found a significant percentage of sperm with double tails, especially in drones obtained in spring (19). In addition, drone sperm from one seasons had different dimensions to sperm from another season. Drone sperm produced in the spring were somewhat longer and also had longer individual structural elements (19). However, according to Morais et al. (36), drone sperm morphometry vary according to rainy and dry seasons. Tarliyah et al. (45) and Power et al. (42) showed high morphological variation in drone sperm. The authors reported a fairly high percentage of sperm with morphological defects, such as flipped tails (19.83%), broken tails (12.75%), double tails (6.42%), and double heads (2.25%) (47). The lack of standardisation of the staining techniques used for evaluation can lead to discrepancies in the results of many comparative studies (22, 30). One solution is computer-assisted semen analysis systems, which limit the subjectivity of morphological analysis (12).

The relatively small number of studies on the morphology and morphometry of drone semen and the lack of established standards regarding staining techniques keep the subject of differential staining of sperm for the purpose of a more precise evaluation relevant. The ideal solution would be the establishment of a staining technique for a given species that would reveal as many structural details as possible while interfering as little as possible with the stained cells. In the case of the staining methods used in the present study, most details were identified using eosin-nigrosin staining. The clearly visible stained structures, especially the midpiece, require further analysis and observations. The modified spermatozoa of insects have great research potential for many scientists. A standard for slide preparation for the evaluation of morphological and morphometric semen parameters should be established, as this would make it possible to compare results between laboratories and increase the value of morphological analysis of sperm for predicting and assessing fertility.