Journal of Apicultural Science 2013, 57(1), 61-65.
The influence of honey bee (Apis mellifera) drone age on volume of semen and viability of spermatozoa.
Krystyna Czekońska, Bożena Chuda-Mickiewicz, Paweł Chorbiński.
A comparison was done of the volume of semen and viability of spermatozoa collected from drones at ages 15, 20, 25 and 30 days. The drones originated from different queens and were reared in different environments. Semen volume was determined by measuring the filled length of a capillary. Percentages of live and dead spermatozoa were determined by SYBR-14/propidium iodide fluorescence staining and flow cytometry. The volume of semen collected from drones ranged from 0.5 to 1.3 µL. The mean volume of semen significantly decreased with drone age. Sperm viability increased significantly with drone age.
Honey bee (Apis mellifera) drones develop from unfertilized eggs. Their development takes 24 days: the egg 3 days, larva 6 days, prepupa and pupa 15 days (Winston, 1987). Spermatogenesis starts at the larval stage and spermiogenesis ends at the pupal stage (Bishop, 1920; Hoage and Kessel, 1968). During the first week of adult life, spermatozoa are transferred from the testes to seminal vesicles, where they are stored until copulation (Snodgrass, 1956). The testes are largest during the pupal stage, and later degenerate (Snodgrass, 1956; Winston, 1987; Page and Peng, 2001).
Drones are not able to copulate immediately after emergence. Copulation and transfer of the spermatozoa from the seminal vesicles to the everted endophallus becomes possible when drones are 10-12 days old (Woyke, 1955; Woyke and Ruttner, 1958, 1976). As drones age, the color and viscosity of their semen changes (Woyke and Jasiński, 1978; Cobey, 2007). The semen of 2-week-old drones is yellowish and fluid; the semen of 4-week-old drones is brown and more viscous. Spermatozoa of old drones are less viable but their motility does not change (Locke and Peng, 1993).
Drones aged 10-21 days are considered most suitable for natural and artificial insemination (Woyke and Ruttner, 1958; Woyke and Jasiński, 1978; Harbo, 1986). Drones younger than 10 days are immature, and the semen of those older than 21 days is too viscous. Queens inseminated with viscous semen transfer fewer spermatozoa in their spermatheca and have difficulty expelling excess semen from their oviducts, which become plugged (Woyke and Jasiński, 1978).
Fertility of the queen depends on the quantity and quality of semen stored in her spermatheca. During natural mating the queen is inseminated by 3-17 drones (Woyke, 1960; Adams et al., 1977; Kraus et al., 2003). To obtain 8 µL of semen for artificial insemination requires the use of 8-80 drones (Bobrzecki, 1968; Prabucki et al., 1992). When there are problems with semen collection, a large number of drones have to be used for artificial insemination (Woyke and Ruttner, 1958; Prabucki et al., 1992; Chuda-Mickiewicz and Prabucki, 1993; Woyke, 2008).
One drone can produce 1.5-1.7 µL of semen (Woyke, 1960). Usually 1.0 µL is collected to a syringe during artificial insemination. This is equivalent to 7.5 million spermatozoa (Woyke, 1960). Amount and quality of the semen is important for the success of both natural and artificial insemination. Queens inseminated with semen of poor quality can be superseded at an earlier age (Woyke and Ruttner, 1976; Cobey 2007). According to Locke and Peng (1993) spermatozoa viability decreases with drone age. Moreover, viability of semen produced by drones stored after emergence in higher temperatures is lower (Bieńkowska et al., 2011). It is not clear, however, if the environment in which drones develop, affect their sperm viability. In this study we compared the volume of semen and the viability of spermatozoa from drones of different ages. The drones originated from different queens and were reared in different environments.
With age, the viscosity of semen increased and its color changed from light cream to dark cream. The mean volume of semen collected from one drone was 0.93 ºL (Tab. 1). The volume of semen collected from drones originating from different queens did not differ significantly (F = 0.611, df = 2, 108, p = 0.545). The mean volume of collected semen decreased with drone age (F = 2.942, df = 3, 108, p = 0.036) (Tab. 2).
The mean viability of sperm was 89.15% (Tab. 1). The viability did not differ between drones originating from different queens (F = 1.100, df = 2, 108, p = 0.335). Sperm viability increased significantly with drone age (F = 6.270, df = 3, 108, p Discussion
The observed reduction of semen volume with drone age, confirms earlier findings (Woyke and JasiÑski, 1978; Locke and Peng, 1993). The viability of spermatozoa did not differ between drones originating from different queens. Locke and Peng (1993) reported reduction of sperm viability from 86% in 14-day-old drones to 81% in 28-day-old drones. In this study we found an increase of sperm viability from 88% in 15-day-old drones to 91% in 30-day-old drones.
One explanation for the increase of sperm viability with age may be related to the gradual maturation of drones. Possibly the drones are able to evert the endophallus and ejaculate before full sexual maturity. In younger drones, the amount of semen remaining in seminal vesicles after ejaculation is higher, and the number of spermatozoa in ejaculate increases with age (Rhodes, 2008). Full maturity of drones may be related not only to more efficient emptying of their seminal vesicles but also to higher sperm viability.
Another possible explanation of increased sperm viability with age might be that drones with higher sperm viability have a higher survival. Drones developing in a better environment might have both longer lives and more viable sperm. In these circumstances, the observed increase in sperm viability could be an effect of poor survival of drones with low sperm viability. In other studies, only 4% of drones survived to the age of 35 days (Rhodes et al., 2011).
The volume of semen collected from one drone decreased with its age. The sperm viability increased with drone age.