Older larvae of honeybee drones are fed with a diet containing pollen. It is not known how pollen deprivation during the larval development of drones might affect their reproductive quality. This study investigated ejaculation ability and semen quality in drones reared in colonies with limited (LP) and unlimited (ULP) access to pollen. Access to pollen was limited by pollen traps. Drone brood rearing was not instantly abandoned in colonies with limited access to pollen. Colonies from the LP group reared drones with smaller mass, which ejaculated in fewer numbers and released smaller amounts of semen. The LP and ULP groups did not differ in semen quality as judged by the concentration, number, and viability of spermatozoa in ejaculate. It was found that access to pollen during larval development directly affects the reproductive quality of drones.
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The studies conducted to date have been concerned mostly with comparing the reproductive value of small drones reared in worker cells and large drones reared in drone cells (Berg et al. 1997; Schlüns et al. 2003; Gençer and Firatli 2005; Couvillon et al. 2010; Gençer and Kahya 2011). Among other findings, it has been shown that larger, heavier drones produce more semen with higher quality spermatozoa and are more successful at mating (Schlüns et al. 2003; Gençer and Firatli 2005; Koeniger et al. 2005; Couvillon et al. 2010; Gençer and Kahya 2011).
It is not known how pollen deprivation during the larval development of drones might affect their reproductive value. The nutrition of drones during their larval development might have effects on their body mass, ability to evert the copulatory apparatus and ejaculate, or the number and viability of their spermatozoa. In this study, we compared ejaculation ability and semen quality in drones reared in colonies with limited or free access to pollen.
The degree of eversion of the copulatory apparatus was assessed by provoking the organ to evert by slightly bending the thorax and pressing it with the fingers, following the method described by Cobey et al. (2013). After eversion of the copulatory apparatus and exudation of semen, the semen was collected in a calibrated microcapillary of known diameter. The length of the microcapillary filled with semen was then measured and converted to volume; 14.3-mm length of the capillary was taken to equal 1-μL semen. The semen was collected using a microcapillary calibrated to 1 μL. The volume of semen was determined by measuring the filled length of the capillary with calipers. Semen volume was determined to 0.1-μL accuracy. The drones were always examined by the same experimenter. In total, 330 drones (183 LP, 147 ULP) were used in assessing readiness to evert the copulatory apparatus and ejaculate.
Spermatozoa concentration per 1-μL semen was determined using the modified method of Woyke (1979). Semen in a known volume was transferred from microcapillaries to tubes which then were filled with saline solution, maintaining a 1:2,000 ratio. After mixing thoroughly, the semen solution was diluted 1:8,000 with distilled water. Spermatozoa were counted in photographs of ten large squares in a Fuschs-Rosenthal chamber taken with a Zeiss Primo Star microscope using Scope Photo software. The number of spermatozoa in 1-μL semen was counted from a total of 35 LP and 37 ULP drone semen samples.
Semen intended for spermatozoa viability assays was diluted in 1,000 μL Kiev buffer. The percentages of live and dead spermatozoa in each sample were determined by SYBR-14/propidium iodide (IP) fluorescence staining with the LIVE/DEAD Sperm Viability Kit (Molecular Probes L-7011). Each 1 μL semen sample was mixed with 5 μL 1:50 diluted Syber-14 and incubated in the dark for 10 min, after which, 2-μL propidium iodide was added and the mixture was incubated for another 10 min. Then, 5-μL incubated semen solution was placed on a glass slide for microscopy. The number of live and dead spermatozoa was counted using an Axio Imager M2 fluorescence microscope. Fifteen fields per preparation were photographed. All samples were prepared and processed in the same way. The obtained images were also analyzed in the same way using AxioVision LE software. The number of live and dead spermatozoa was counted from a total of 19 LP and 15 ULP semen samples.
Semen volume was estimated using 244 portions of semen from both LP and ULP drones (Table I). On average, significantly less semen 0.2 μL (drone on average) was collected from LP drones than from ULP drones.
The spermatozoa concentration per 1-μL semen did not differ significantly between the LP and ULP drones (Table I) nor did the number of spermatozoa differ. The semen from the LP and ULP drones showed similar levels of spermatozoa viability, with no significant difference (Table I).
Drones reared in the LP colonies everted the copulatory apparatus but ejaculated less frequently and released smaller amounts of semen than the ULP drones. Possibly, the LP drones at 15 days of age had no semen or were sexually immature and not yet able to ejaculate so that they released small amounts of semen if any. Perhaps drones from colonies with limited access to pollen do not participate in reproduction and die quickly or have a shorter reproductive period. Our results could help explain the reported large differences in the reproductive success of drones from different colonies (Kraus et al. 2003; Rueppell et al. 2006; Couvillon et al. 2010), and differences in the number of drones needed to collect one dose of semen for queen bee insemination (Woyke 1960, 2010; Chuda-Mickiewicz and Prabucki 1993; Rhodes et al. 2011).
The range of volume of semen we measured from the drones is comparable to that reported in other studies (0.1 to 1.8 μL) (Woyke 1960; Chuda-Mickiewicz and Prabucki 1993; Schlüns et al. 2003; Rhodes et al. 2011; Gençer and Kahya 2011; Czekońska et al. 2013a), but the concentration of spermatozoa was lower. Regardless of the treatment group, in 1-μL semen, we estimated more than two million fewer spermatozoa than the reports of Woyke (1960), Mackensen (1964), Bobrzecki (1968), and Gençer and Kahya (2011), who obtained 7.5, 7.64, 7.36, and 7.26 million, respectively. Chuda-Mickiewicz and Prabucki (1993) gave figures closer to ours: 4.9 to 6.1 million spermatozoa per 1 μL. There were no differences in spermatozoa concentration between the LP and ULP drones.
We found that honeybee colonies do not immediately abandon drone brood rearing when access to pollen is limited. Workers rear a brood that produces drones with lower mass, ejaculating less frequently, and giving smaller volumes of semen. The LP colonies did not rear brood with lower semen quality, however, as judged by the spermatozoa concentration and by the number and viability of spermatozoa in the ejaculate.
In this work, 32 % of the drones reared in nutritionally poorer conditions and 20 % of those fed normally had problems in everting the copulatory apparatus and ejaculating. Drones reared under poorer nutrition probably mature later and delay their nuptial flights. The briefer reproductive period lowers their chance of mating with the queen, reducing the likelihood of delivering semen to her.
Cincinnati Zoo and Botanical Garden Center for Conservation and Research on Endangered Wildlife published article using OptiXcell for the cryopreservation of rhinoceros semen in the August 2019 issue of Cryobiology.
Milk-based semen diluents have proven to be practical and effective in protecting sperm cells during storage prior to artificial insemination; however, milk components may not be at optimal concentrations, and some may even be detrimental to sperm cells.
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