Cryobiology 69 (2014) 236–242
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A successful new approach to honeybee semen cryopreservation q Jakob Wegener a,⇑, Tanja May b, Günter Kamp b, Kaspar Bienefeld a a b
Institute for Bee Research Hohen Neuendorf, Friedrich-Engels-Strasse 32, 16540 Hohen Neuendorf, Germany AMP-Lab GmbH, University Campus, Becherweg 9-11, 55099 Mainz, Germany
a r t i c l e
i n f o
Article history: Received 18 March 2014 Accepted 21 July 2014 Available online 1 August 2014 Keywords: Apis mellifera Sperm Slow freezing Dialysis Artificial insemination
a b s t r a c t Honeybee biodiversity is under massive threat, and improved methods for gamete cryopreservation could be a precious tool for both the in situ- and ex situ-conservation of subspecies and ecotypes. Recent cryoprotocols for drone semen have improved the viability and fertility of frozen-thawed semen by using increased diluent:semen-ratios, but there is still much room for progress. As semen cryopreserved after dilution often appeared hyperactive, we speculated that the disruption of sperm–sperm interactions during dilution and cryopreservation could reduce the fertile lifespan of the cells. We therefore developed protocols to reduce admixture, or abolish it altogether by dialyzing semen against a hypertonic solution of cryoprotectant. Additionally, we tested methods to reduce the cryoprotectant concentration after thawing. Insemination of queens with semen cryopreserved after dialysis yielded 49%, 59% and 79% female (=stemming from fertilized eggs) pupae in three separate experiments, and the numbers of sperm found in the spermathecae of the queens were significantly higher than those previously reported. Post-thaw dilution and reconcentration of semen for cryoprotectant removal reduced fertility, but sizeable proportions of female brood were still produced. Workers stemming from cryopreserved semen did not differ from bees stemming from untreated semen with regard to indicators of fluctuating asymmetry, but were slightly heavier. Cryopreservation after dialysis tended to increase the proportion of cells with DNA-nicks, as measured by the TUNEL-assay, but this increase appears small when compared to the baseline variations of this indicator. Overall, we conclude that cryoprotectant-addition through dialysis can improve the quality of cryopreserved drone semen. Testing of offspring for vitality and genetic integrity should continue. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Two factors that limit the genetic progress in honeybee breeding are the short annual period of semen availability (typically 3–5 months in temperate climates) and the long duration of performance testing in relation to the total lifespan of breeding queens. Cryopreservation of drone semen could ease these restrictions, and enable crosses between partners that are separated by space and/or time. In a situation where many subspecies and ecotypes of Apis mellifera are threatened by the loss of genetic identity
q Statement of funding: This study was supported by funds of the German Ministry for Food, Agriculture and Consumer Security (BMELV) through the intermediary of the Federal Office for Agriculture and Food (BLE), within the framework of the program for innovation (FKZ 2813500408 and 2813500508). Material used was financed with the help of the European Fund for Regional Development (EFRE; Application No. 80137041). ⇑ Corresponding author. Fax: +49 3303 293840. E-mail addresses: [email protected] (J. Wegener), [email protected] (T. May), [email protected] (G. Kamp), [email protected] (K. Bienefeld).
http://dx.doi.org/10.1016/j.cryobiol.2014.07.011 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
[2,12,14], semen cryopreservation could also be a valuable tool for both in situ- and ex situ-conservation of genotypes. First attempts to cryopreserve drone semen have been made during the late 1970s and early 1980s [3,4,9,13]. Some of these studies succeeded in preserving a high degree of motility after thawing [9], but fertility showed out to be strongly limited [4,9]. Recently, drone semen cryopreservation has received renewed interest [7,8,21,25,27]. Taylor et al. [21] have shown that higher dilution rates (>1:3 semen:diluent) yield slightly higher semen viability. Hopkins et al. (2012) [8] have used this approach in an insemination experiment. They succeeded in producing queens that are offspring of cryopreserved semen, and inseminating these with semen of the same origin to yield animals that show a heightened degree of genetic relatedness to the cryopreserved genotype. Despite these advancements, fertility of cryopreserved drone semen is still very low. Two of the main indicators of sperm fertility in A. mellifera are the proportion of female brood that inseminated queens produce (because in Hymenoptera, only fertilized eggs develop into females, whereas lack of fertilization results in the development of males), and the number of sperm that reach
J. Wegener et al. / Cryobiology 69 (2014) 236–242
the storage organ of the queen (because this number is a strong predictor of the functional longevity of queens). Already in 1984, Kaftanoglu and Peng reached an average of 56% female brood and 290,000 sperm in spermathecae, using semen samples that were pre-selected for high motility after thawing [9]. Hopkins et al. [8] reached an average of 50% (range 0–100%) female brood without pre-selection of semen samples. Queens inseminated with frozen-thawed semen disappeared from the hives within 2 months after insemination, suggesting that sperm numbers inside the thecae were probably low, or queens were negatively affected by components of the semen diluent. The most successful cryoprotocols published so far use slow freezing with MeSO as the sole cryoprotectant [4,8,9]. MeSO is suspected of causing genetic damage in honey bee sperm [5]. It is also known that insemination with MeSO-containing semen can cause a reduction of sperm numbers reaching the storage organ of queens [25]. In the present study, we therefore tested a method to partially remove MeSO after thawing by introducing a washing/centrifugation step. Additionally, we tested protocols that reduce the level of semen dilution. Current protocols involve the dilution of semen through the admixture of extenders for the addition of cryoprotectants. Honeybee semen has a high cell density of around 7 million/lL, and cells form masses with parallel flagellae. Dilution causes a strong increase of motility in honey bee sperm [10]. It is known from other insects that ejaculated sperm are embedded in a kind of extracellular matrix, whose destruction facilitates activation [15]. In many vertebrates, longevity of sperm cells is generally greatly reduced after activation [16]. Kaftanoglu and Peng [9] have found that the motility of frozen-thawed semen was sometimes higher than that of fresh semen, although the fertility after insemination proved to be poor. This matched our own observations with protocols involving the use of a diluent for the addition of cryoprotectant. We therefore hypothesized that reducing the level of dilution could be beneficial for survival of sperm after thawing, and devised cryoprotocols that minimize this effect by either maximizing the semen: diluent ratio, or by dialyzing semen against a hypertonic solution of cryoprotectant. The three objectives of this study were to increase the fertility of frozen-thawed semen by reducing the level of semen dilution, to reduce the exposure of semen and queens to MeSO, and to test the suitability of a dilution-free protocol for longer-term preservation of fertility and genetic integrity. We first performed two experiments in which different protocols aiming to reduce semen dilution as well as exposure of semen and queens to MeSO were compared by inseminating queens and observing their brood nests as well as the contents of their spermathecae. The protocol yielding the largest proportion of female brood was then further used in two additional experiments, one testing its ability to preserve semen fertility for a longer period of time (9 months), the second evaluating the impact of cryostorage on genetic integrity by using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. First data on the fertile period of queens inseminated with frozen-thawed semen, as well as the effects of semen cryopreservation on morphometric traits of worker offspring, are also reported.
Materials and methods Production and handling of semen and queens For each experiment, capped drone brood of the subspecies A.m. carnica was transferred into strong, queenless colonies for further rearing and sexual maturation. Semen was collected at 20–22 (experiment 1), 35–37 (experiment 3) or 40–45 days (experiment 2) post-emergence. Ejaculation was triggered manually by exerting
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pressure on the thorax, and semen was taken up into glass capillaries using an insemination syringe back-filled with Bee Sperm Solution (BSS) [27], containing 0.01% penicillin-GK and 0.02% streptomycin sulfate. The average volume of semen collected per drone was close to 1 lL in all experiments. A new capillary was started every 40–80 lL. After collection, semen was stored for a maximum of 2 days at 12–15 °C before use (exception: see experiment 4). Queens for the insemination experiments were reared in queenless colonies according to standard techniques [17]. Groups of sister queens were used for each experiment. Upon emergence, they were held in micro-colonies of approximately 2000 bees that were equipped with excluder grids in front of the hive entrances in order to prevent natural mating. Queens were narcotized with CO2 for 7 min 1 day before insemination. Insemination was performed under CO2-narcosis using a Schley-apparatus (Peter Schley, Germany), and queens were re-introduced into their micro-colonies. The age of queens at insemination was 12–13 days (experiments 1 and 3) or 7–8 days (experiment 3). Experiment 1: Cryoprotocols with reduced semen dilution/MeSOremoval Cryoprotocol A – admixture of diluent with reduced dilution ratio A total of 110 lL of semen from two capillaries (representing drones from two different mother queens) were pooled in a 0.5 mL centrifuge tube. A solution of 50% MeSO in double-distilled water was added at the ratio of 1 lL diluent to every 4 lL of undiluted semen (dilution 1:1.25; final concentration of MeSO in the semen/diluent-mixture 10% v/v). In order to limit osmotic shock, diluent was added using the calibrated capillary and plunger of a microcapillary pipette (Capilettor; Selzer, Germany) in the way of a microliter syringe. The calibrated capillary was connected with a piece of silicone tubing to a second, thinner capillary (interior diameter 0.32 mm, outer diameter 0.4 mm; Hilgenberg, Germany). Diluent was added very slowly through the thinner capillary, while permanently stirring the semen with the capillary. After 10 min of equilibration, portions of 9 lL of the semen/diluent-mixture were filled into 4 cm-long pieces of polyethylene tubing (outer diameter 1.8 mm, inner diameter 1.0 mm; Karl Roth, Germany) with the help of a direct displacement pipette (MicroMan, Gilson, Germany), leaving approximately 1 cm of air at each end of the tubes. The ends were heat-sealed. The samples were placed into cryotubes that had been transpierced to allow rapid access of liquid nitrogen. They were frozen in a controlled-rate freezer (IceCube, SyLab, Austria). They were held at 20 °C for 5 min, then cooled at 3°/min for 20 min, followed by plunging into liquid nitrogen (cooling rate after [9]). After 48 h of cryo-storage, samples were thawed by immersion into a water bath at 35 °C for 10 s. The semen was taken up directly into the insemination syringe, using small pieces of silicone tubing as adaptors. Fourteen queens were inseminated, each receiving 8 lL of diluted semen. Cryoprotocol B – MeSO-loading via semen dialysis Two micro-dialysis chambers (AMP-Lab, Germany; chamber volume approx. 100 lL) were incubated in double-distilled water for 30 min. Semen (70–80 lL) was added into each chamber directly from the capillaries in which it had been stored. Chambers were placed into glass vials containing 20 mL of a 21% v/v solution of MeSO in BSS and dialysed for 32 min at 22.5 °C on a microplate shaker. The dialyzed semen was portioned, packed and frozen as described above. It was thawed in a bath of paraffin oil at 35 °C for 15 s, because preliminary experiments had shown that this yielded better results than thawing in a water bath. Thirteen queens were inseminated with the thawed semen. As semen was visibly concentrated after dialysis, each queen only received a dose of 6 lL.
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Cryoprotocol C – partial removal of MeSO after thawing Semen was cryopreserved and thawed as with the dialysis-protocol. After thawing, each portion was suspended in 152 lL of BSS and taken up into 200 lL standard micropipette tips. The tips were heat-sealed at the thinner ends and centrifuged at 1300g for 12 min [26]. After removal of the supernatant, they were cut open and semen taken up into the insemination syringe. Fourteen queens were inseminated in this way. Given that reconcentration of sperm after centrifugation was incomplete [26], the insemination volume was increased to 8.5 lL to reach sperm numbers similar to those in the other treatments. In addition to the experimental queens, three control queens were inseminated with 7 lL unfrozen semen of the same genetic origin as that used in the cryo-experiments. In order to keep the duration of unfrozen storage of the semen comparable to that of semen used for the cryo-protocols, controls had to be inseminated 2 days (i.e. the duration of cryo-storage of the experimental semen) earlier than the remaining queens. Analysis of sperm from spermathecae Thirty-nine days after their insemination, those queens that had not been introduced into full-sized colonies for overwintering (see below) were dissected to investigate the number, motility and membrane integrity (‘‘viability’’) of sperm inside their spermathecae. The thecae were dissected in 20 lL of K+-buffer [26], and the sperm taken up into 100 lL of the same liquid. Aliquots of these suspensions were used for the determination of the maximum proportion of cells in which motility could be induced in an activating buffer (K+; details in [26]), as well as for membrane permeability using the fluorochromes propidium iodide and Hoechst 33342 [11,25,26]. Morphologic investigation of offspring from cryopreserved semen Eighteen to 19 days after the first eggs had appeared in a microcolony, all capped brood was removed and sexed according to the type of cell cap (flat or domed). The brood combs were incubated at 35 °C/50–70% relative humidity until no more adults emerged. Every day, the emerging workers were collected. Their fresh weight was determined, and they were frozen at 20 °C. Among them, thirty worker bees per cryoprotocol were chosen at random and dissected for morphometrical analysis. Two measures of fluctuating asymmetry were used as indicators of developmental stress, the difference between the numbers of ovarioles in the left and right ovary and the difference between the centroid sizes of the two forewings [19,22]. Overwintering of queens inseminated with cryopreserved semen To test the long-term storability of frozen-thawed semen inside the queen’s spermatheca, the three animals with the highest proportion of female offspring of each treatment group (but not the control) were introduced into full-sized colonies on October 12 and overwintered. The surviving queens were recovered on April 26. Motility of sperm inside their spermathecae was measured, and a comb of their brood was used to determine the proportion of female offspring. Semen of drones stemming from the same two mother queens was used for each of the three cryoprotocols tested as well as for the controls.
Cryoprotocol D involved dialysis against a reduced concentration of MeSO (13.5% v/v). Duration of the dialysis-step was increased to 150 min, and the process was performed at a higher temperature (34.5 °C). The samples were thawed for 10 s in water instead of paraffin. Six queens were inseminated with the thawed semen (6–7 lL/queen). Cryoprotocol E was equal to the dialysis protocol of experiment 1, except that the semen was diluted 1:2 with BSS (containing 0.01% penicillin-GK and 0.02% streptomycin sulfate) post-thawing. The blend was stirred only slightly in order to minimize dissolution of sperm clusters. It was taken up into the insemination syringe and used to inseminate three queens (10 lL/ queen). Six queens inseminated with semen cryopreserved according to Protocol B (dialysis-protocol of experiment 1; 6–7 lL/animal) were used as controls. In contrast to experiments 1 and 2, semen was not frozen in single-queen portions, but in one single portion for all queens of each treatment. This allowed for a reduction of the occurrence of small air bubbles in the semen. Small subsamples of the thawed semen were diluted approximately 1:1500 in K+ and used for the determination of post-thaw motility. The proportions of female brood as well as the motility and number of sperm from the spermathecae were measured as in experiment 1. One queen from the control group and one from the group that had received the protocol E-semen were used for a test of queen rearing from larvae fathered by cryopreserved semen. Fourteen larvae from each of the two queens were placed inside a queenless rearing colony according to standard techniques [17]. They were transferred to an incubator after cell capping, and replaced by another 15 + 15 larvae. Emerging queens were visually checked for exterior morphological abnormalities. Larvae from experiment 2 were also used for two tests of queen-rearing from larvae stemming from cryopreserved semen. For the first, two rearing colonies were formed, and one received 30 larvae from one of the queens from experiment 3, while the other received 30 larvae from a normal colony headed by a naturally-mated queen. In the second, larvae from queens having received protocol B and E-semen in experiment 2 were compared in a single starter colony. A first set of 14 + 14 larvae was introduced. After the accepted cells had been capped, they were transferred to an incubator to continue development, while the rearing colony received a second set of 15 + 15 larvae that was treated in the same way as the first. Experiment 3: Longer-term cryostorage of semen preserved via dialysis A total of 120 lL of semen (from drones that were all sons of the same queen) were frozen following protocol B of experiment 1 (dialysis against BSS), and transported to the cryobank of the Fraunhofer-Institute for Biomedical Technology (Sulzbach, Germany) in a dry shipper using express postal service. After 9 months of storage, they were transported back and used to inseminate seven queens. The proportion of female brood produced by these animals was determined as described. An additional sample of the stored semen was thawed, diluted 1:1500 in K+ and used for direct determination of motility.
Experiment 2: Variants of the dialysis-protocol
Experiment 4: Quantification of DNA-damage caused by freezing/ thawing of semen
The dialysis protocol was modulated to reduce osmotic stress experienced by the cells during dialysis, and/or MeSO-exposure of sperm and queens.
For the quantification of DNA-breakage through freezing/ thawing, three semen samples from drones stemming from different mothers were subdivided, and aliquots were either frozen
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according to protocol B or kept as unfrozen controls. Two were frozen according to protocol A and B, while the third was kept as an untreated control. The samples had been stored at 12–15 °C for 25, 39 and 120 days, but still contained >90% motile cells before cryopreservation. Nuclei containing DNA-nicks were identified using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The TUNEL-assay was performed using the respective kit from Roche (Germany). The methodology followed the manufacturer’s manual, with a few changes. Semen samples were diluted 1:500 with BSS-buffer, mixed gently at room temperature, smeared on poly-lysin coated cover glasses (18 18 mm) and air dried. For each sample seven cover glasses were prepared – five for sample evaluation, one for a positive control (pretreatment with DNAse) and another for a negative control (no incubation with TUNEL solution but only with fluorescein dUTP). After 1 h of fixation in freshly prepared fixation solution (17 °C; 4% paraformaldehyde in PBS (pH 7,4), the cover slips were gently rinsed with PBS. They were incubated for exactly 2 min with freshly prepared permeabilizing solution (0.1% Triton X-100; 0.1% sodium citrate). During the incubation, the samples lay in prechilled Petri dishes on ice, in order to ensure a temperature of about 2 °C. Then the coverslips were washed twice with PBS and air-dried. After adding 50 lL TUNEL solution per coverslip, the samples were incubated in a humidified atmosphere for 60 min at 37 °C in the dark. The TUNEL solution contained the terminal deoxynucleotidyl transferase (Tdt) and the fluorescein dUTP. After incubation with the TUNEL solution, the samples were rinsed three times with PBS and then analyzed under a fluorescence microscope (excitation wavelength 450–490 nm; emission wavelength 515– 565 nm). Only sperm with DNA strand breaks exhibited a distinct green fluorescence. Images were taken and saved for later analysis using Photoshop CS5 and Microsoft Office Excel.
Results Experiment 1: Cryoprotocols with reduced dilution/MeSO-removal All but five of the 44 queens initiated egglaying within 16 days after insemination. Of these five, three started to lay later (17–28 days after insemination), while one was accidentally killed. The last queen was found dead after 27 days. She had received semen containing visible impurities, as noted during insemination. The three queens inseminated with untreated semen produced 100% female brood, as compared to 45.7% (29.2–77.8), 47.5% (25.6–72.5) and 27.0% (0.5–65.3) in queens receiving semen cryopreserved following protocols A (admixture of cryoprotectant), B (dialysis) and C (dialysis + centrifugation), respectively (fig. 1A). Protocols A and B formed a homogenous subgroup (adjusted P = 0.73), differing from protocol C (dialysis + centrifugation) and the fresh semen (P < 0.05). With 3.6 million, the number of sperm in queens receiving fresh semen was 5.1 times higher than with the best cryoprotocol (dialysis, 0.7 million; P < 0.05; fig. 1B). Protocols B and C formed a homogenous subgroup (adjusted P = 0.23), as did protocols C and D (adjusted P = 0.17). The motility and membrane impermeability to propidium iodide were more similar between the treatments (fig. 1C), with only the differences between fresh semen and each of the cryoprotocols being significant (P < 0.05). Table 1 summarizes the measurements performed on the worker progeny of the inseminated queens. Weights at emergence were slightly different between the semen treatments (F = 4.3, P = 0.005), with workers produced with untreated semen being on overage 6.0 and 6.8 mg lighter than those fathered by sperm preserved according either the admixture or dialysis-protocols (P = 0.04 and 0.004, respectively). No significant differences occurred with regard to the asymmetries of forewing size (X2 = 6.48; P = 0.09) or ovariole numbers (F = 0.89, P = 047). Of the nine queens introduced into full-sized colonies, one (from the group receiving centrifuged semen) escaped during introduction and was lost. One queen from each of the other two groups disappeared during the winter, so that two queens per treatment group survived into the spring and could be identified by the numbered plastic tags they had received after insemination. The percentages of motile cells among the sperm recuperated from their thecae were 7.4 and 7.1 (admixture), 0.0 and 50.7 (dialysis), and 8.8 and 5.7 (dialysis + centrifugation). Brood samples from
100
a
5
A
80 60 ab
ab
40
b
20
Number of sperm in spermatheca [million]
Proportion of female brood [%]
Comparisons between cryoprotocols regarding parameters of sperm number and quality as well as proportions of female brood were made using the Kruskal–Wallis test, followed by homogenous subgroup determination [20]. Measurements performed on offspring of cryopreserved semen were compared using either the Kruskal–Wallis test or one-factorial ANOVA, depending on the distribution of data. IBM-SPSS 19.0 was used for all analyses [20].
B
a
4 3 2 ab
1
bc c
0
0
a
1 2 b
80
ab
C
1,2
2 b
Motility Intactness of cell membrane
60 40 20 0
Semen treatment
dialysis + centrifugation
dialysis
admixture
fresh semen
Semen treatment
dialysis + centrifugation
admixture
dialysis
fresh semen
dialysis + centrifugation
dialysis
admixture
fresh semen
Semen treatment
100
Proportion of sperm [%]
Statistical analysis
Fig. 1. Fertility, motility and membrane integrity of semen cryopreserved with reduced-dilution protocols. Different letters or numbers indicate significant differences between semen treatment variants (P < 0.05). Fig. 1C refers to semen recovered from the spermathecae of inseminated queens. ‘‘Motility’’ means the proportion of sperm showing any type of active movement. ‘‘Intactness of cell membrane’’ means impermeability to propidium iodide.
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Table 1 Onset of egglaying and properties of workers produced after insemination with cryopreserved semen. Unfrozen semen
Cryoprotocol Admixture
Dialysis
Dialysis + centrifug.
11.4 ± 8.1 (n = 14)
10.2 ± 3.1 (n = 12)
9.6 ± 3.0 (n = 14)
Onset of egglaying (days after insemin.) Weight of workers at emergence (mg) Asymmetry: ovariole numbers
15.8 ± 11.0 (n = 3) 119.1 ± 16.9 (n = 105) 2.1 ± 1.5 (n = 30)
125.1 ± 14.8 (n = 85) 1.3 ± 1.0 (n = 28)
125.9 ± 15.4 (n = 142) 1.9 ± 1.5 (n = 29)
124.1 ± 15.6 (n = 114) 1.5 ± 1.9 (n = 28)
Asymmetry: wing nervature
6.0 ± 6.5 (n = 30)
7.4 ± 6.3 (n = 29)
7.1 ± 7.7 (n = 29)
9.4 ± 10.9 (n = 28
a
b
b
ab
Test used
Value of test statistic
P
n.d.
–
–
ANOVA
F = 4.3
0.005
Kruskal– Wallis ANOVA
X2 = 6.48
0.090
F = 0.89
0.447
n.d. = not determined. a, b, ab Different letters indicate significant differences between treatments (P < 0.05).
a
60 40 20
a
1.5 1.0
a
0.5
21.0%, diluted after thawing
b
ab
80 60
a
40 20 0
Semen treatment
C
13.5% MeSO
0.0
21.0% MeSO
Semen treatment
2.0
13.5% MeSO
21.0%, diluted after thawing
21.0% MeSO
13.5% MeSO
0
Number of sperm in spermatheca [million]
ab
100
21.0%, diluted after thawing
b
80
B
Proportion of motile sperm [%]
a
2.5
A
21.0% MeSO
Proportion of female brood [%]
100
Semen treatment
Fig. 2. Fertility and motility of semen cryopreserved with different variants of the protocol for cryoprotectant addition through dialysis. Different letters indicate significant differences between semen treatment variants (P < 0.05). Fig. 2C refers to semen recovered from the spermathecae of inseminated queens. ‘‘Motile sperm’’ means cells that show any type of active movement.
these queens contained 95%) female brood. Additionally, the use of cryopreserved semen often seemed to lead to brood nests with an increased number of empty cells. Although this ‘‘patchiness’’ is hard to quantify, it likely results from the removal of deficient brood by worker bees, and indicates that not all fertilizations by cryopreserved sperm are successful. On the other hand, of a total of 25 queens inseminated with semen cryopreserved after dialysis against 21% MeSO (protocol B; sum of experiments 1, 2 and 3), 22 produced at least some female offspring. This proportion shows that the method repeatably yields a level of fertility that should be sufficient for the use of cryopreserved semen in breeding programs. Cryoprotectant-addition through dialysis also allowed an increase in the number of sperm reaching the storage organ of queens. This parameter is critical for the duration of the queen’s fertile period (reviewed in [1]). The numbers found in experiment 1 and the control of experiment 2 represent more than a doubling of those reported by Kaftanoglu and Peng (290,000; mean calculated from data in [9]), which appear to be the highest values published so far. Average sperm numbers in the thecae of queens from experiment 1 were likely reduced by the fact that the three animals showing the highest proportion of female brood were used for the overwintering trial, and therefore could not be dissected. The fact that one queen inseminated with dialysed semen still produced a sizeable proportion of fertilized eggs after overwintering could mean that cryopreservation after dialysis indeed allows the preservation not only of short-term fertility, but also of in vivo-storability of drone semen. This conclusion cannot be drawn with certainty, since it is based on results from one queen only, while the second overwintering animal of the same treatment group produced exclusively unfertilized eggs. A higher proportion of female brood and higher sperm-numbers inside the spermathecae of queens were obtained in the controls of experiment 2, as compared to the other two groups of queens receiving dialysed semen in experiments 1 and 3. This could be due to the different ways of portioning the semen. The singlequeen portions used in experiments 1 and 2 might have led to a higher number of air bubbles, frequently forming at the two ends of the semen column, and to an increased contact with oxygen. Oxidative damage of sperm suspected to be a limiting factor for the quality of frozen-thawed honey bee semen [21]. Another factor involved could be drone age – significantly older males were used in experiment 2 than in the other two experiments. Slight dilution of the semen post-thawing (protocol E of experiment 2) tended to further increase sperm numbers in the spermatheca, but as only three queens were inseminated with semen treated in this way, this point clearly needs further validation. If confirmed, the positive effect of dilution on sperm migration to the spermatheca may be linked to the fact that motility is required for this process [18], and that motility of bee sperm is known to be reduced in hypertonic environments and increased by dilution [10,23]. Two factors could explain the positive results obtained with cryopreservation after dialysis. Firstly, the intracellular concentration of osmolytes might have been increased, favoring vitrification of the cells. Honey bee seminal plasma has an osmolarity of only 325 mOsm [24], and optimum survival of diluted sperm suspensions is generally seen at around 450 mOsm [26], meaning that
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the dialysis buffer of protocol B was strongly hypertonic. Semen volume after dialysis was clearly reduced, although volumetric measurements are difficult because an unknown fraction of the original semen sticks to the dialysis chamber and cannot be recovered. Secondly, addition of the cryoprotectant through dialysis instead of admixture may partially prevent the disruption of sperm–sperm interactions, which otherwise may lead to precocious activation and reduce the capacity for long-term survival of sperm in the spermatheca. Sperm are present in ejaculated semen as well as in semen from the spermathecae of naturally-mated queens in the form of dense bundles, and the destruction of these bundles through stirring and dilution may be deleterious. Osanai and Baccetti [15] have shown that the proteolytic dissociation of lepidopteran and orthopteran sperm bundles is an important step in the activation of the cells. In fact, the trigger for using dialysis to add cryoprotectants was the observation that semen cryopreserved after diluent admixture always showed maximum motility almost immediately after thawing and suspension in an activating medium (K+), whereas fresh semen only reached this state after 30–60 min. Although not exactly measured, sperm that had been cryopreserved after dialysis appeared to show intermediate activation times. The videos of freshly thawed and diluted semen provided as additional materials 1 and 2 seem to support the hypothesis that protocol B (dialysis against 21% MeSO) helps to preserve sperm clusters, showing that with this protocol, clusters of sperm remain, whereas sperm from the other treatments of experiment 1 appear mostly disordered. The second objective of this study was to reduce exposure of sperm and/or queens to cryoprotectants. Independently of cryopreservation, MeSO-treatment of sperm has been reported to cause infertility in a low percentage of female progeny [6]. It has also been shown that 10% MeSO in unfrozen semen reduce the number of cells reaching the spermatheca [25]. Attempts to replace MeSO as cryoprotectant for drone semen have so far failed [21]. We therefore developed a methodology for the centrifugation of drone semen [26], in order to partially remove MeSO by washing the cells after thawing. The washing protocol was tested with unfrozen semen and led to only a marginal reduction of the numbers of sperm reaching the spermatheca [26]. As experiment 1 shows, this is also true for frozen-thawed sperm. The motility and viability of the cells recovered from the spermathecae however was significantly lower than without the washing step, which likely explains the lower fertility of the centrifuged sperm. Nevertheless, all but one of the queens receiving the centrifuged semen produced female offspring, so MeSO-removal through cell washing may be an option, should future experiments show that the concerns regarding genetic damage by MeSO are justified. Protocol D of experiment 2, using a strongly reduced MeSO-concentration in the dialysis buffer but a longer duration of dialysis, was a second attempt at reducing MeSO-exposure and/or hypertonic stress. We do not know whether a reduction of the concentration of MeSO in the semen was actually achieved, because due to the difficulty of measuring MeSO chromatographically, levels inside the dialysed semen were not determined for either protocol. It is likely however that protocol D led to less severe dehydration of semen, since only a slight reduction of volume was observed. Although post-thaw motility achieved with the reduced MeSO-protocol was excellent, fertility was greatly lowered in comparison to protocol B, indicating that sperm were sublethally damaged. We do not know whether this was due to a higher amount of intracellular water or to the efflux of important components from the seminal plasma during the long period of dialysis. The third goal of this study was to test the usefulness of the dialysis-protocol (protocol B) for cryobanking. It seems encouraging that frozen semen could be transported by postal services, stored in a professional cryobank for 9 months and used to produce
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female offspring. Because of the concerns regarding possible transgenerational effects of MeSO [5], and because influences of semen cryopreservation on genetic integrity and performance of offspring are critical to the usefulness of the technique for breeding, we measured DNA-breakage in frozen-thawed sperm, as well as some morphological traits in workers produced from fertilizations with cryopreserved semen. The slight increase of the frequency of DNA-nicks after cryopreservation appears to be negligible when compared to the great amount of variation between the unfrozen samples, which is likely linked to either natural causes or conditions during liquid storage. The measurements of fluctuating asymmetry performed on workers produced from frozen-thawed sperm from all treatments of experiment 1 showed no significant deviations from the values found in controls from inseminations with untreated semen. Fluctuating asymmetry is seen as an indicator of developmental instability [19]. In the honeybee, it has been linked, for example, to the occurrence of colony collapse disorder (CCD; [22]). The slight increase in weight, observed in workers from the cryo-treatments, could possibly be a result of the fact that queens from these treatments produced slightly smaller brood nests, so that available resources per larva may have been higher. The prolonged developmental period of two of the queens stemming from protocol B-semen could possibly hint developmental troubles, but this problem did not occur in the repetition of the queen rearing experiment. Overall, our results seem to suggest that semen cryopreservation, even using MeSO, is probably of no great consequence for the quality of offspring produced, but that more detailed analyses on this point are desirable. Honeybee biodiversity is currently under massive threat from the introgression of foreign genes into endemic populations [2]. The beekeeping industry, which is mostly to blame for this, is itself under pressure to come up with lines that are tolerant to severe introduced parasitoses and the as yet unknown causes of colony collapse disorder [22]. Cryobanking of bee semen could be a tool to help preserve bee diversity, and fasten genetic progress. The method of dialysing sperm for the addition of cryoprotectants presented here repeatably leads to frozen-thawed semen with a fertility that should be sufficient for the production of offspring queens. This is the prerequisite for use in breeding programs. The fact that queens appear to remain fertile for longer periods than with other published protocols [8] should facilitate the use of cryopreserved semen in breeding. An additional advantage over earlier protocols could be the fact that no addition of egg yolk is needed. As a natural product, yolk may represent sanitary risks and reduce repeatability of protocols [8]. Although our results are insufficient to conclude with certainty that the method has no influence on genetic integrity and offspring performance, we hope that they will represent a contribution to solving some of the honeybee’s many problems. Acknowledgments We are very grateful to Anja Rogge for semen collection and expert apicultural assistance, and to Karin Müller for technical advice. The beekeepers of the Bee Research Institute helped by providing bees and queens. This study was supported by funds of the German Ministry for Food, Agriculture and Consumer Security (BMELV) through the intermediary of the Federal Office for Agriculture and Food (BLE), within the framework of the program for innovation (FKZ 2813500408 and 2813500508). Material used was financed with the help of the European Fund for Regional Development (EFRE; Application No. 80137041).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cryobiol.2014. 07.011. References [1] S.W. Cobey, Comparison studies of instrumentally inseminated and naturally mated honey bee queens and factors affecting their performance, Apidologie 38 (2007) 390–410. [2] P. De la Rùa, R. Jaffé, R. Dall’Olio, I. Munoz, J. Serrano, Biodiversity, conservation and current threats to European honeybees, Apidologie 40 (2009) 263–284. [3] J. Harbo, Survival of honey bee spermatozoa in liquid nitrogen, Ann. Entomol. Soc. Am. 70 (1977) 257–258. [4] J. Harbo, Survival of honey bee (Hymenoptera: Apidae) spermatozoa after 2 years in liquid nitrogen ( 196 °C), Ann. Entomol. Soc. Am. 76 (1983) 890– 891. [5] J. Harbo, Sterility in honey bees caused by dimethyl sulfoxide, J. Hered. 77 (1986) 129–130. [6] J.R. Harbo, Sterility in honey bees caused by dimethyl sulfoxide, J. Hered. 77 (1986) 129–130. [7] B.K. Hopkins, C. Herr, Factors affecting the successful cryopreservation of honey bee (Apis mellifera) spermatozoa, Apidologie 41 (2010) 548–556. [8] B.K. Hopkins, C. Herr, W.S. Sheppard, Sequential generations of honey bee (Apis mellifera) queens produced using cryopreserved semen, Reprod. Fertil. Dev. 24 (2012) 1079–1083. [9] O. Kaftanoglu, Y.C. Peng, Preservation of honeybee spermatozoa in liquid nitrogen, J. Apic. Res. 23 (1984) 157–163. [10] Y. Lensky, H. Schindler, Motility and reversible inactivation of honeybee spermatozoa in vivo and in vitro, Annales de l’Abeille 10 (1967) 5–16. [11] S.J. Locke, Y.-S. Peng, N.L. Cross, A supravital staining technique for honey bee spermatozoa, Physiol. Entomol. 15 (1990). [12] M.D. Meixner, C. Costa, P. Kryger, F. Hatjina, M. Bouga, E. Ivanova, R. Büchler, Conserving diversity and vitality for honeybee breeding, J. Apic. Res. 49 (2010) 85–92. [13] A.N. Melnichenko, I.L. Vavilov, Long term storage of drone sperm by freezing in liquid nitrogen, Apimondia, Prague, 1975, pp. 311–314. [14] A. Oleksa, I. Chybicki, A. Tofilski, J. Burczyk, Nuclear and mitochondrial patterns of introgression into native dark bees (Apis mellifera) in Poland, J. Apic. Res. 50 (2011) 129–160. [15] M. Osanai, B. Baccetti, Two-step acquisition of motility by insect spermatozoa, Experientia 49 (1993) 593–595. [16] S. Pitnick, M.F. Wolfner, S.S. Suarez, Ejaculate-female and sperm-female interactions, in: T.R. Birkhead, D.J. Hosken, S. Pitnick (Eds.), Sperm Biology – An Evolutionary Perspective, Academic Press, Burlington, San Diego, Oxford, 2009, pp. 247–304. [17] F. Ruttner, Zuchttechnik und Zuchtauslese bei der Biene, Ehrenworth, Munich, 1996. [18] F. Ruttner, G. Koeniger, Filling of spermatheca of honeybee queen – active migration or passive transport of spermatozoa, Zeitschrift für vergleichende Physiologie 72 (1971) 411–422. [19] S.S. Schneider, L.J. Leamy, L.A. Lewis, G. DeGrandi-Hoffman, The influence of hybridization between African and European honeybees, Apis mellifera, on asymmetries in wing size and shape, Evolution 57 (2003) 2350–2364. [20] SPSS, IBM SPSS Statistics 19 core system user’s guide, SPSS, 2010. [21] M.A. Taylor, E. Guzmán-Novoa, N. Morfin, M.M. Buhr, Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios, Theriogenology 72 (2009) 149– 159. [22] D. vanEngelsdorp, J.D. Evans, C. Saegerman, C. Mullin, E. Haubruge, et al., Colony collapse disorder: a descriptive study, PLoS ONE 4 (2009) e6481. http:// dx.doi.org/10.1371/journal.pone.0006481. [23] L.R. Verma, An ionic basis for a possible mechanism of sperm survival in the spermatheca of the queen honey bee (Apis mellifera L.), Comp. Biochem. Physiol. A Comp. Physiol. 44 (1972) 1345–1351. [24] L.R. Verma, Osmotic analysis of honeybee (Apis mellifera L.) semen and haemolymph, Am. Bee J. 113 (1973) 412. [25] J. Wegener, K. Bienefeld, Toxicity of cryoprotectants to honey bee semen and queens, Theriogenology 77 (2012) 600–607. [26] J. Wegener, T. May, G. Kamp, K. Bienefeld, New methods and media for the centrifugation of honey bee (Hymenoptera: Apidae) drone semen, J. Econ. Entomol. 107 (2014) 47–53. [27] J. Wegener, T. May, U. Knollmann, G. Kamp, K. Bienefeld, In vivo validation of in vitro quality tests for cryopreserved honeybee semen, Cryobiology 65 (2012) 126–131.
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
A successful new approach to honeybee semen cryopreservation q Jakob Wegener a,⇑, Tanja May b, Günter Kamp b, Kaspar Bienefeld a a b
Institute for Bee Research Hohen Neuendorf, Friedrich-Engels-Strasse 32, 16540 Hohen Neuendorf, Germany AMP-Lab GmbH, University Campus, Becherweg 9-11, 55099 Mainz, Germany
a r t i c l e
i n f o
Article history: Received 18 March 2014 Accepted 21 July 2014 Available online 1 August 2014 Keywords: Apis mellifera Sperm Slow freezing Dialysis Artificial insemination
a b s t r a c t Honeybee biodiversity is under massive threat, and improved methods for gamete cryopreservation could be a precious tool for both the in situ- and ex situ-conservation of subspecies and ecotypes. Recent cryoprotocols for drone semen have improved the viability and fertility of frozen-thawed semen by using increased diluent:semen-ratios, but there is still much room for progress. As semen cryopreserved after dilution often appeared hyperactive, we speculated that the disruption of sperm–sperm interactions during dilution and cryopreservation could reduce the fertile lifespan of the cells. We therefore developed protocols to reduce admixture, or abolish it altogether by dialyzing semen against a hypertonic solution of cryoprotectant. Additionally, we tested methods to reduce the cryoprotectant concentration after thawing. Insemination of queens with semen cryopreserved after dialysis yielded 49%, 59% and 79% female (=stemming from fertilized eggs) pupae in three separate experiments, and the numbers of sperm found in the spermathecae of the queens were significantly higher than those previously reported. Post-thaw dilution and reconcentration of semen for cryoprotectant removal reduced fertility, but sizeable proportions of female brood were still produced. Workers stemming from cryopreserved semen did not differ from bees stemming from untreated semen with regard to indicators of fluctuating asymmetry, but were slightly heavier. Cryopreservation after dialysis tended to increase the proportion of cells with DNA-nicks, as measured by the TUNEL-assay, but this increase appears small when compared to the baseline variations of this indicator. Overall, we conclude that cryoprotectant-addition through dialysis can improve the quality of cryopreserved drone semen. Testing of offspring for vitality and genetic integrity should continue. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Two factors that limit the genetic progress in honeybee breeding are the short annual period of semen availability (typically 3–5 months in temperate climates) and the long duration of performance testing in relation to the total lifespan of breeding queens. Cryopreservation of drone semen could ease these restrictions, and enable crosses between partners that are separated by space and/or time. In a situation where many subspecies and ecotypes of Apis mellifera are threatened by the loss of genetic identity
q Statement of funding: This study was supported by funds of the German Ministry for Food, Agriculture and Consumer Security (BMELV) through the intermediary of the Federal Office for Agriculture and Food (BLE), within the framework of the program for innovation (FKZ 2813500408 and 2813500508). Material used was financed with the help of the European Fund for Regional Development (EFRE; Application No. 80137041). ⇑ Corresponding author. Fax: +49 3303 293840. E-mail addresses: [email protected] (J. Wegener), [email protected] (T. May), [email protected] (G. Kamp), [email protected] (K. Bienefeld).
http://dx.doi.org/10.1016/j.cryobiol.2014.07.011 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
[2,12,14], semen cryopreservation could also be a valuable tool for both in situ- and ex situ-conservation of genotypes. First attempts to cryopreserve drone semen have been made during the late 1970s and early 1980s [3,4,9,13]. Some of these studies succeeded in preserving a high degree of motility after thawing [9], but fertility showed out to be strongly limited [4,9]. Recently, drone semen cryopreservation has received renewed interest [7,8,21,25,27]. Taylor et al. [21] have shown that higher dilution rates (>1:3 semen:diluent) yield slightly higher semen viability. Hopkins et al. (2012) [8] have used this approach in an insemination experiment. They succeeded in producing queens that are offspring of cryopreserved semen, and inseminating these with semen of the same origin to yield animals that show a heightened degree of genetic relatedness to the cryopreserved genotype. Despite these advancements, fertility of cryopreserved drone semen is still very low. Two of the main indicators of sperm fertility in A. mellifera are the proportion of female brood that inseminated queens produce (because in Hymenoptera, only fertilized eggs develop into females, whereas lack of fertilization results in the development of males), and the number of sperm that reach
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the storage organ of the queen (because this number is a strong predictor of the functional longevity of queens). Already in 1984, Kaftanoglu and Peng reached an average of 56% female brood and 290,000 sperm in spermathecae, using semen samples that were pre-selected for high motility after thawing [9]. Hopkins et al. [8] reached an average of 50% (range 0–100%) female brood without pre-selection of semen samples. Queens inseminated with frozen-thawed semen disappeared from the hives within 2 months after insemination, suggesting that sperm numbers inside the thecae were probably low, or queens were negatively affected by components of the semen diluent. The most successful cryoprotocols published so far use slow freezing with MeSO as the sole cryoprotectant [4,8,9]. MeSO is suspected of causing genetic damage in honey bee sperm [5]. It is also known that insemination with MeSO-containing semen can cause a reduction of sperm numbers reaching the storage organ of queens [25]. In the present study, we therefore tested a method to partially remove MeSO after thawing by introducing a washing/centrifugation step. Additionally, we tested protocols that reduce the level of semen dilution. Current protocols involve the dilution of semen through the admixture of extenders for the addition of cryoprotectants. Honeybee semen has a high cell density of around 7 million/lL, and cells form masses with parallel flagellae. Dilution causes a strong increase of motility in honey bee sperm [10]. It is known from other insects that ejaculated sperm are embedded in a kind of extracellular matrix, whose destruction facilitates activation [15]. In many vertebrates, longevity of sperm cells is generally greatly reduced after activation [16]. Kaftanoglu and Peng [9] have found that the motility of frozen-thawed semen was sometimes higher than that of fresh semen, although the fertility after insemination proved to be poor. This matched our own observations with protocols involving the use of a diluent for the addition of cryoprotectant. We therefore hypothesized that reducing the level of dilution could be beneficial for survival of sperm after thawing, and devised cryoprotocols that minimize this effect by either maximizing the semen: diluent ratio, or by dialyzing semen against a hypertonic solution of cryoprotectant. The three objectives of this study were to increase the fertility of frozen-thawed semen by reducing the level of semen dilution, to reduce the exposure of semen and queens to MeSO, and to test the suitability of a dilution-free protocol for longer-term preservation of fertility and genetic integrity. We first performed two experiments in which different protocols aiming to reduce semen dilution as well as exposure of semen and queens to MeSO were compared by inseminating queens and observing their brood nests as well as the contents of their spermathecae. The protocol yielding the largest proportion of female brood was then further used in two additional experiments, one testing its ability to preserve semen fertility for a longer period of time (9 months), the second evaluating the impact of cryostorage on genetic integrity by using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. First data on the fertile period of queens inseminated with frozen-thawed semen, as well as the effects of semen cryopreservation on morphometric traits of worker offspring, are also reported.
Materials and methods Production and handling of semen and queens For each experiment, capped drone brood of the subspecies A.m. carnica was transferred into strong, queenless colonies for further rearing and sexual maturation. Semen was collected at 20–22 (experiment 1), 35–37 (experiment 3) or 40–45 days (experiment 2) post-emergence. Ejaculation was triggered manually by exerting
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pressure on the thorax, and semen was taken up into glass capillaries using an insemination syringe back-filled with Bee Sperm Solution (BSS) [27], containing 0.01% penicillin-GK and 0.02% streptomycin sulfate. The average volume of semen collected per drone was close to 1 lL in all experiments. A new capillary was started every 40–80 lL. After collection, semen was stored for a maximum of 2 days at 12–15 °C before use (exception: see experiment 4). Queens for the insemination experiments were reared in queenless colonies according to standard techniques [17]. Groups of sister queens were used for each experiment. Upon emergence, they were held in micro-colonies of approximately 2000 bees that were equipped with excluder grids in front of the hive entrances in order to prevent natural mating. Queens were narcotized with CO2 for 7 min 1 day before insemination. Insemination was performed under CO2-narcosis using a Schley-apparatus (Peter Schley, Germany), and queens were re-introduced into their micro-colonies. The age of queens at insemination was 12–13 days (experiments 1 and 3) or 7–8 days (experiment 3). Experiment 1: Cryoprotocols with reduced semen dilution/MeSOremoval Cryoprotocol A – admixture of diluent with reduced dilution ratio A total of 110 lL of semen from two capillaries (representing drones from two different mother queens) were pooled in a 0.5 mL centrifuge tube. A solution of 50% MeSO in double-distilled water was added at the ratio of 1 lL diluent to every 4 lL of undiluted semen (dilution 1:1.25; final concentration of MeSO in the semen/diluent-mixture 10% v/v). In order to limit osmotic shock, diluent was added using the calibrated capillary and plunger of a microcapillary pipette (Capilettor; Selzer, Germany) in the way of a microliter syringe. The calibrated capillary was connected with a piece of silicone tubing to a second, thinner capillary (interior diameter 0.32 mm, outer diameter 0.4 mm; Hilgenberg, Germany). Diluent was added very slowly through the thinner capillary, while permanently stirring the semen with the capillary. After 10 min of equilibration, portions of 9 lL of the semen/diluent-mixture were filled into 4 cm-long pieces of polyethylene tubing (outer diameter 1.8 mm, inner diameter 1.0 mm; Karl Roth, Germany) with the help of a direct displacement pipette (MicroMan, Gilson, Germany), leaving approximately 1 cm of air at each end of the tubes. The ends were heat-sealed. The samples were placed into cryotubes that had been transpierced to allow rapid access of liquid nitrogen. They were frozen in a controlled-rate freezer (IceCube, SyLab, Austria). They were held at 20 °C for 5 min, then cooled at 3°/min for 20 min, followed by plunging into liquid nitrogen (cooling rate after [9]). After 48 h of cryo-storage, samples were thawed by immersion into a water bath at 35 °C for 10 s. The semen was taken up directly into the insemination syringe, using small pieces of silicone tubing as adaptors. Fourteen queens were inseminated, each receiving 8 lL of diluted semen. Cryoprotocol B – MeSO-loading via semen dialysis Two micro-dialysis chambers (AMP-Lab, Germany; chamber volume approx. 100 lL) were incubated in double-distilled water for 30 min. Semen (70–80 lL) was added into each chamber directly from the capillaries in which it had been stored. Chambers were placed into glass vials containing 20 mL of a 21% v/v solution of MeSO in BSS and dialysed for 32 min at 22.5 °C on a microplate shaker. The dialyzed semen was portioned, packed and frozen as described above. It was thawed in a bath of paraffin oil at 35 °C for 15 s, because preliminary experiments had shown that this yielded better results than thawing in a water bath. Thirteen queens were inseminated with the thawed semen. As semen was visibly concentrated after dialysis, each queen only received a dose of 6 lL.
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Cryoprotocol C – partial removal of MeSO after thawing Semen was cryopreserved and thawed as with the dialysis-protocol. After thawing, each portion was suspended in 152 lL of BSS and taken up into 200 lL standard micropipette tips. The tips were heat-sealed at the thinner ends and centrifuged at 1300g for 12 min [26]. After removal of the supernatant, they were cut open and semen taken up into the insemination syringe. Fourteen queens were inseminated in this way. Given that reconcentration of sperm after centrifugation was incomplete [26], the insemination volume was increased to 8.5 lL to reach sperm numbers similar to those in the other treatments. In addition to the experimental queens, three control queens were inseminated with 7 lL unfrozen semen of the same genetic origin as that used in the cryo-experiments. In order to keep the duration of unfrozen storage of the semen comparable to that of semen used for the cryo-protocols, controls had to be inseminated 2 days (i.e. the duration of cryo-storage of the experimental semen) earlier than the remaining queens. Analysis of sperm from spermathecae Thirty-nine days after their insemination, those queens that had not been introduced into full-sized colonies for overwintering (see below) were dissected to investigate the number, motility and membrane integrity (‘‘viability’’) of sperm inside their spermathecae. The thecae were dissected in 20 lL of K+-buffer [26], and the sperm taken up into 100 lL of the same liquid. Aliquots of these suspensions were used for the determination of the maximum proportion of cells in which motility could be induced in an activating buffer (K+; details in [26]), as well as for membrane permeability using the fluorochromes propidium iodide and Hoechst 33342 [11,25,26]. Morphologic investigation of offspring from cryopreserved semen Eighteen to 19 days after the first eggs had appeared in a microcolony, all capped brood was removed and sexed according to the type of cell cap (flat or domed). The brood combs were incubated at 35 °C/50–70% relative humidity until no more adults emerged. Every day, the emerging workers were collected. Their fresh weight was determined, and they were frozen at 20 °C. Among them, thirty worker bees per cryoprotocol were chosen at random and dissected for morphometrical analysis. Two measures of fluctuating asymmetry were used as indicators of developmental stress, the difference between the numbers of ovarioles in the left and right ovary and the difference between the centroid sizes of the two forewings [19,22]. Overwintering of queens inseminated with cryopreserved semen To test the long-term storability of frozen-thawed semen inside the queen’s spermatheca, the three animals with the highest proportion of female offspring of each treatment group (but not the control) were introduced into full-sized colonies on October 12 and overwintered. The surviving queens were recovered on April 26. Motility of sperm inside their spermathecae was measured, and a comb of their brood was used to determine the proportion of female offspring. Semen of drones stemming from the same two mother queens was used for each of the three cryoprotocols tested as well as for the controls.
Cryoprotocol D involved dialysis against a reduced concentration of MeSO (13.5% v/v). Duration of the dialysis-step was increased to 150 min, and the process was performed at a higher temperature (34.5 °C). The samples were thawed for 10 s in water instead of paraffin. Six queens were inseminated with the thawed semen (6–7 lL/queen). Cryoprotocol E was equal to the dialysis protocol of experiment 1, except that the semen was diluted 1:2 with BSS (containing 0.01% penicillin-GK and 0.02% streptomycin sulfate) post-thawing. The blend was stirred only slightly in order to minimize dissolution of sperm clusters. It was taken up into the insemination syringe and used to inseminate three queens (10 lL/ queen). Six queens inseminated with semen cryopreserved according to Protocol B (dialysis-protocol of experiment 1; 6–7 lL/animal) were used as controls. In contrast to experiments 1 and 2, semen was not frozen in single-queen portions, but in one single portion for all queens of each treatment. This allowed for a reduction of the occurrence of small air bubbles in the semen. Small subsamples of the thawed semen were diluted approximately 1:1500 in K+ and used for the determination of post-thaw motility. The proportions of female brood as well as the motility and number of sperm from the spermathecae were measured as in experiment 1. One queen from the control group and one from the group that had received the protocol E-semen were used for a test of queen rearing from larvae fathered by cryopreserved semen. Fourteen larvae from each of the two queens were placed inside a queenless rearing colony according to standard techniques [17]. They were transferred to an incubator after cell capping, and replaced by another 15 + 15 larvae. Emerging queens were visually checked for exterior morphological abnormalities. Larvae from experiment 2 were also used for two tests of queen-rearing from larvae stemming from cryopreserved semen. For the first, two rearing colonies were formed, and one received 30 larvae from one of the queens from experiment 3, while the other received 30 larvae from a normal colony headed by a naturally-mated queen. In the second, larvae from queens having received protocol B and E-semen in experiment 2 were compared in a single starter colony. A first set of 14 + 14 larvae was introduced. After the accepted cells had been capped, they were transferred to an incubator to continue development, while the rearing colony received a second set of 15 + 15 larvae that was treated in the same way as the first. Experiment 3: Longer-term cryostorage of semen preserved via dialysis A total of 120 lL of semen (from drones that were all sons of the same queen) were frozen following protocol B of experiment 1 (dialysis against BSS), and transported to the cryobank of the Fraunhofer-Institute for Biomedical Technology (Sulzbach, Germany) in a dry shipper using express postal service. After 9 months of storage, they were transported back and used to inseminate seven queens. The proportion of female brood produced by these animals was determined as described. An additional sample of the stored semen was thawed, diluted 1:1500 in K+ and used for direct determination of motility.
Experiment 2: Variants of the dialysis-protocol
Experiment 4: Quantification of DNA-damage caused by freezing/ thawing of semen
The dialysis protocol was modulated to reduce osmotic stress experienced by the cells during dialysis, and/or MeSO-exposure of sperm and queens.
For the quantification of DNA-breakage through freezing/ thawing, three semen samples from drones stemming from different mothers were subdivided, and aliquots were either frozen
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according to protocol B or kept as unfrozen controls. Two were frozen according to protocol A and B, while the third was kept as an untreated control. The samples had been stored at 12–15 °C for 25, 39 and 120 days, but still contained >90% motile cells before cryopreservation. Nuclei containing DNA-nicks were identified using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The TUNEL-assay was performed using the respective kit from Roche (Germany). The methodology followed the manufacturer’s manual, with a few changes. Semen samples were diluted 1:500 with BSS-buffer, mixed gently at room temperature, smeared on poly-lysin coated cover glasses (18 18 mm) and air dried. For each sample seven cover glasses were prepared – five for sample evaluation, one for a positive control (pretreatment with DNAse) and another for a negative control (no incubation with TUNEL solution but only with fluorescein dUTP). After 1 h of fixation in freshly prepared fixation solution (17 °C; 4% paraformaldehyde in PBS (pH 7,4), the cover slips were gently rinsed with PBS. They were incubated for exactly 2 min with freshly prepared permeabilizing solution (0.1% Triton X-100; 0.1% sodium citrate). During the incubation, the samples lay in prechilled Petri dishes on ice, in order to ensure a temperature of about 2 °C. Then the coverslips were washed twice with PBS and air-dried. After adding 50 lL TUNEL solution per coverslip, the samples were incubated in a humidified atmosphere for 60 min at 37 °C in the dark. The TUNEL solution contained the terminal deoxynucleotidyl transferase (Tdt) and the fluorescein dUTP. After incubation with the TUNEL solution, the samples were rinsed three times with PBS and then analyzed under a fluorescence microscope (excitation wavelength 450–490 nm; emission wavelength 515– 565 nm). Only sperm with DNA strand breaks exhibited a distinct green fluorescence. Images were taken and saved for later analysis using Photoshop CS5 and Microsoft Office Excel.
Results Experiment 1: Cryoprotocols with reduced dilution/MeSO-removal All but five of the 44 queens initiated egglaying within 16 days after insemination. Of these five, three started to lay later (17–28 days after insemination), while one was accidentally killed. The last queen was found dead after 27 days. She had received semen containing visible impurities, as noted during insemination. The three queens inseminated with untreated semen produced 100% female brood, as compared to 45.7% (29.2–77.8), 47.5% (25.6–72.5) and 27.0% (0.5–65.3) in queens receiving semen cryopreserved following protocols A (admixture of cryoprotectant), B (dialysis) and C (dialysis + centrifugation), respectively (fig. 1A). Protocols A and B formed a homogenous subgroup (adjusted P = 0.73), differing from protocol C (dialysis + centrifugation) and the fresh semen (P < 0.05). With 3.6 million, the number of sperm in queens receiving fresh semen was 5.1 times higher than with the best cryoprotocol (dialysis, 0.7 million; P < 0.05; fig. 1B). Protocols B and C formed a homogenous subgroup (adjusted P = 0.23), as did protocols C and D (adjusted P = 0.17). The motility and membrane impermeability to propidium iodide were more similar between the treatments (fig. 1C), with only the differences between fresh semen and each of the cryoprotocols being significant (P < 0.05). Table 1 summarizes the measurements performed on the worker progeny of the inseminated queens. Weights at emergence were slightly different between the semen treatments (F = 4.3, P = 0.005), with workers produced with untreated semen being on overage 6.0 and 6.8 mg lighter than those fathered by sperm preserved according either the admixture or dialysis-protocols (P = 0.04 and 0.004, respectively). No significant differences occurred with regard to the asymmetries of forewing size (X2 = 6.48; P = 0.09) or ovariole numbers (F = 0.89, P = 047). Of the nine queens introduced into full-sized colonies, one (from the group receiving centrifuged semen) escaped during introduction and was lost. One queen from each of the other two groups disappeared during the winter, so that two queens per treatment group survived into the spring and could be identified by the numbered plastic tags they had received after insemination. The percentages of motile cells among the sperm recuperated from their thecae were 7.4 and 7.1 (admixture), 0.0 and 50.7 (dialysis), and 8.8 and 5.7 (dialysis + centrifugation). Brood samples from
100
a
5
A
80 60 ab
ab
40
b
20
Number of sperm in spermatheca [million]
Proportion of female brood [%]
Comparisons between cryoprotocols regarding parameters of sperm number and quality as well as proportions of female brood were made using the Kruskal–Wallis test, followed by homogenous subgroup determination [20]. Measurements performed on offspring of cryopreserved semen were compared using either the Kruskal–Wallis test or one-factorial ANOVA, depending on the distribution of data. IBM-SPSS 19.0 was used for all analyses [20].
B
a
4 3 2 ab
1
bc c
0
0
a
1 2 b
80
ab
C
1,2
2 b
Motility Intactness of cell membrane
60 40 20 0
Semen treatment
dialysis + centrifugation
dialysis
admixture
fresh semen
Semen treatment
dialysis + centrifugation
admixture
dialysis
fresh semen
dialysis + centrifugation
dialysis
admixture
fresh semen
Semen treatment
100
Proportion of sperm [%]
Statistical analysis
Fig. 1. Fertility, motility and membrane integrity of semen cryopreserved with reduced-dilution protocols. Different letters or numbers indicate significant differences between semen treatment variants (P < 0.05). Fig. 1C refers to semen recovered from the spermathecae of inseminated queens. ‘‘Motility’’ means the proportion of sperm showing any type of active movement. ‘‘Intactness of cell membrane’’ means impermeability to propidium iodide.
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Table 1 Onset of egglaying and properties of workers produced after insemination with cryopreserved semen. Unfrozen semen
Cryoprotocol Admixture
Dialysis
Dialysis + centrifug.
11.4 ± 8.1 (n = 14)
10.2 ± 3.1 (n = 12)
9.6 ± 3.0 (n = 14)
Onset of egglaying (days after insemin.) Weight of workers at emergence (mg) Asymmetry: ovariole numbers
15.8 ± 11.0 (n = 3) 119.1 ± 16.9 (n = 105) 2.1 ± 1.5 (n = 30)
125.1 ± 14.8 (n = 85) 1.3 ± 1.0 (n = 28)
125.9 ± 15.4 (n = 142) 1.9 ± 1.5 (n = 29)
124.1 ± 15.6 (n = 114) 1.5 ± 1.9 (n = 28)
Asymmetry: wing nervature
6.0 ± 6.5 (n = 30)
7.4 ± 6.3 (n = 29)
7.1 ± 7.7 (n = 29)
9.4 ± 10.9 (n = 28
a
b
b
ab
Test used
Value of test statistic
P
n.d.
–
–
ANOVA
F = 4.3
0.005
Kruskal– Wallis ANOVA
X2 = 6.48
0.090
F = 0.89
0.447
n.d. = not determined. a, b, ab Different letters indicate significant differences between treatments (P < 0.05).
a
60 40 20
a
1.5 1.0
a
0.5
21.0%, diluted after thawing
b
ab
80 60
a
40 20 0
Semen treatment
C
13.5% MeSO
0.0
21.0% MeSO
Semen treatment
2.0
13.5% MeSO
21.0%, diluted after thawing
21.0% MeSO
13.5% MeSO
0
Number of sperm in spermatheca [million]
ab
100
21.0%, diluted after thawing
b
80
B
Proportion of motile sperm [%]
a
2.5
A
21.0% MeSO
Proportion of female brood [%]
100
Semen treatment
Fig. 2. Fertility and motility of semen cryopreserved with different variants of the protocol for cryoprotectant addition through dialysis. Different letters indicate significant differences between semen treatment variants (P < 0.05). Fig. 2C refers to semen recovered from the spermathecae of inseminated queens. ‘‘Motile sperm’’ means cells that show any type of active movement.
these queens contained 95%) female brood. Additionally, the use of cryopreserved semen often seemed to lead to brood nests with an increased number of empty cells. Although this ‘‘patchiness’’ is hard to quantify, it likely results from the removal of deficient brood by worker bees, and indicates that not all fertilizations by cryopreserved sperm are successful. On the other hand, of a total of 25 queens inseminated with semen cryopreserved after dialysis against 21% MeSO (protocol B; sum of experiments 1, 2 and 3), 22 produced at least some female offspring. This proportion shows that the method repeatably yields a level of fertility that should be sufficient for the use of cryopreserved semen in breeding programs. Cryoprotectant-addition through dialysis also allowed an increase in the number of sperm reaching the storage organ of queens. This parameter is critical for the duration of the queen’s fertile period (reviewed in [1]). The numbers found in experiment 1 and the control of experiment 2 represent more than a doubling of those reported by Kaftanoglu and Peng (290,000; mean calculated from data in [9]), which appear to be the highest values published so far. Average sperm numbers in the thecae of queens from experiment 1 were likely reduced by the fact that the three animals showing the highest proportion of female brood were used for the overwintering trial, and therefore could not be dissected. The fact that one queen inseminated with dialysed semen still produced a sizeable proportion of fertilized eggs after overwintering could mean that cryopreservation after dialysis indeed allows the preservation not only of short-term fertility, but also of in vivo-storability of drone semen. This conclusion cannot be drawn with certainty, since it is based on results from one queen only, while the second overwintering animal of the same treatment group produced exclusively unfertilized eggs. A higher proportion of female brood and higher sperm-numbers inside the spermathecae of queens were obtained in the controls of experiment 2, as compared to the other two groups of queens receiving dialysed semen in experiments 1 and 3. This could be due to the different ways of portioning the semen. The singlequeen portions used in experiments 1 and 2 might have led to a higher number of air bubbles, frequently forming at the two ends of the semen column, and to an increased contact with oxygen. Oxidative damage of sperm suspected to be a limiting factor for the quality of frozen-thawed honey bee semen [21]. Another factor involved could be drone age – significantly older males were used in experiment 2 than in the other two experiments. Slight dilution of the semen post-thawing (protocol E of experiment 2) tended to further increase sperm numbers in the spermatheca, but as only three queens were inseminated with semen treated in this way, this point clearly needs further validation. If confirmed, the positive effect of dilution on sperm migration to the spermatheca may be linked to the fact that motility is required for this process [18], and that motility of bee sperm is known to be reduced in hypertonic environments and increased by dilution [10,23]. Two factors could explain the positive results obtained with cryopreservation after dialysis. Firstly, the intracellular concentration of osmolytes might have been increased, favoring vitrification of the cells. Honey bee seminal plasma has an osmolarity of only 325 mOsm [24], and optimum survival of diluted sperm suspensions is generally seen at around 450 mOsm [26], meaning that
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the dialysis buffer of protocol B was strongly hypertonic. Semen volume after dialysis was clearly reduced, although volumetric measurements are difficult because an unknown fraction of the original semen sticks to the dialysis chamber and cannot be recovered. Secondly, addition of the cryoprotectant through dialysis instead of admixture may partially prevent the disruption of sperm–sperm interactions, which otherwise may lead to precocious activation and reduce the capacity for long-term survival of sperm in the spermatheca. Sperm are present in ejaculated semen as well as in semen from the spermathecae of naturally-mated queens in the form of dense bundles, and the destruction of these bundles through stirring and dilution may be deleterious. Osanai and Baccetti [15] have shown that the proteolytic dissociation of lepidopteran and orthopteran sperm bundles is an important step in the activation of the cells. In fact, the trigger for using dialysis to add cryoprotectants was the observation that semen cryopreserved after diluent admixture always showed maximum motility almost immediately after thawing and suspension in an activating medium (K+), whereas fresh semen only reached this state after 30–60 min. Although not exactly measured, sperm that had been cryopreserved after dialysis appeared to show intermediate activation times. The videos of freshly thawed and diluted semen provided as additional materials 1 and 2 seem to support the hypothesis that protocol B (dialysis against 21% MeSO) helps to preserve sperm clusters, showing that with this protocol, clusters of sperm remain, whereas sperm from the other treatments of experiment 1 appear mostly disordered. The second objective of this study was to reduce exposure of sperm and/or queens to cryoprotectants. Independently of cryopreservation, MeSO-treatment of sperm has been reported to cause infertility in a low percentage of female progeny [6]. It has also been shown that 10% MeSO in unfrozen semen reduce the number of cells reaching the spermatheca [25]. Attempts to replace MeSO as cryoprotectant for drone semen have so far failed [21]. We therefore developed a methodology for the centrifugation of drone semen [26], in order to partially remove MeSO by washing the cells after thawing. The washing protocol was tested with unfrozen semen and led to only a marginal reduction of the numbers of sperm reaching the spermatheca [26]. As experiment 1 shows, this is also true for frozen-thawed sperm. The motility and viability of the cells recovered from the spermathecae however was significantly lower than without the washing step, which likely explains the lower fertility of the centrifuged sperm. Nevertheless, all but one of the queens receiving the centrifuged semen produced female offspring, so MeSO-removal through cell washing may be an option, should future experiments show that the concerns regarding genetic damage by MeSO are justified. Protocol D of experiment 2, using a strongly reduced MeSO-concentration in the dialysis buffer but a longer duration of dialysis, was a second attempt at reducing MeSO-exposure and/or hypertonic stress. We do not know whether a reduction of the concentration of MeSO in the semen was actually achieved, because due to the difficulty of measuring MeSO chromatographically, levels inside the dialysed semen were not determined for either protocol. It is likely however that protocol D led to less severe dehydration of semen, since only a slight reduction of volume was observed. Although post-thaw motility achieved with the reduced MeSO-protocol was excellent, fertility was greatly lowered in comparison to protocol B, indicating that sperm were sublethally damaged. We do not know whether this was due to a higher amount of intracellular water or to the efflux of important components from the seminal plasma during the long period of dialysis. The third goal of this study was to test the usefulness of the dialysis-protocol (protocol B) for cryobanking. It seems encouraging that frozen semen could be transported by postal services, stored in a professional cryobank for 9 months and used to produce
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female offspring. Because of the concerns regarding possible transgenerational effects of MeSO [5], and because influences of semen cryopreservation on genetic integrity and performance of offspring are critical to the usefulness of the technique for breeding, we measured DNA-breakage in frozen-thawed sperm, as well as some morphological traits in workers produced from fertilizations with cryopreserved semen. The slight increase of the frequency of DNA-nicks after cryopreservation appears to be negligible when compared to the great amount of variation between the unfrozen samples, which is likely linked to either natural causes or conditions during liquid storage. The measurements of fluctuating asymmetry performed on workers produced from frozen-thawed sperm from all treatments of experiment 1 showed no significant deviations from the values found in controls from inseminations with untreated semen. Fluctuating asymmetry is seen as an indicator of developmental instability [19]. In the honeybee, it has been linked, for example, to the occurrence of colony collapse disorder (CCD; [22]). The slight increase in weight, observed in workers from the cryo-treatments, could possibly be a result of the fact that queens from these treatments produced slightly smaller brood nests, so that available resources per larva may have been higher. The prolonged developmental period of two of the queens stemming from protocol B-semen could possibly hint developmental troubles, but this problem did not occur in the repetition of the queen rearing experiment. Overall, our results seem to suggest that semen cryopreservation, even using MeSO, is probably of no great consequence for the quality of offspring produced, but that more detailed analyses on this point are desirable. Honeybee biodiversity is currently under massive threat from the introgression of foreign genes into endemic populations [2]. The beekeeping industry, which is mostly to blame for this, is itself under pressure to come up with lines that are tolerant to severe introduced parasitoses and the as yet unknown causes of colony collapse disorder [22]. Cryobanking of bee semen could be a tool to help preserve bee diversity, and fasten genetic progress. The method of dialysing sperm for the addition of cryoprotectants presented here repeatably leads to frozen-thawed semen with a fertility that should be sufficient for the production of offspring queens. This is the prerequisite for use in breeding programs. The fact that queens appear to remain fertile for longer periods than with other published protocols [8] should facilitate the use of cryopreserved semen in breeding. An additional advantage over earlier protocols could be the fact that no addition of egg yolk is needed. As a natural product, yolk may represent sanitary risks and reduce repeatability of protocols [8]. Although our results are insufficient to conclude with certainty that the method has no influence on genetic integrity and offspring performance, we hope that they will represent a contribution to solving some of the honeybee’s many problems. Acknowledgments We are very grateful to Anja Rogge for semen collection and expert apicultural assistance, and to Karin Müller for technical advice. The beekeepers of the Bee Research Institute helped by providing bees and queens. This study was supported by funds of the German Ministry for Food, Agriculture and Consumer Security (BMELV) through the intermediary of the Federal Office for Agriculture and Food (BLE), within the framework of the program for innovation (FKZ 2813500408 and 2813500508). Material used was financed with the help of the European Fund for Regional Development (EFRE; Application No. 80137041).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cryobiol.2014. 07.011. References [1] S.W. Cobey, Comparison studies of instrumentally inseminated and naturally mated honey bee queens and factors affecting their performance, Apidologie 38 (2007) 390–410. [2] P. De la Rùa, R. Jaffé, R. Dall’Olio, I. Munoz, J. Serrano, Biodiversity, conservation and current threats to European honeybees, Apidologie 40 (2009) 263–284. [3] J. Harbo, Survival of honey bee spermatozoa in liquid nitrogen, Ann. Entomol. Soc. Am. 70 (1977) 257–258. [4] J. Harbo, Survival of honey bee (Hymenoptera: Apidae) spermatozoa after 2 years in liquid nitrogen ( 196 °C), Ann. Entomol. Soc. Am. 76 (1983) 890– 891. [5] J. Harbo, Sterility in honey bees caused by dimethyl sulfoxide, J. Hered. 77 (1986) 129–130. [6] J.R. Harbo, Sterility in honey bees caused by dimethyl sulfoxide, J. Hered. 77 (1986) 129–130. [7] B.K. Hopkins, C. Herr, Factors affecting the successful cryopreservation of honey bee (Apis mellifera) spermatozoa, Apidologie 41 (2010) 548–556. [8] B.K. Hopkins, C. Herr, W.S. Sheppard, Sequential generations of honey bee (Apis mellifera) queens produced using cryopreserved semen, Reprod. Fertil. Dev. 24 (2012) 1079–1083. [9] O. Kaftanoglu, Y.C. Peng, Preservation of honeybee spermatozoa in liquid nitrogen, J. Apic. Res. 23 (1984) 157–163. [10] Y. Lensky, H. Schindler, Motility and reversible inactivation of honeybee spermatozoa in vivo and in vitro, Annales de l’Abeille 10 (1967) 5–16. [11] S.J. Locke, Y.-S. Peng, N.L. Cross, A supravital staining technique for honey bee spermatozoa, Physiol. Entomol. 15 (1990). [12] M.D. Meixner, C. Costa, P. Kryger, F. Hatjina, M. Bouga, E. Ivanova, R. Büchler, Conserving diversity and vitality for honeybee breeding, J. Apic. Res. 49 (2010) 85–92. [13] A.N. Melnichenko, I.L. Vavilov, Long term storage of drone sperm by freezing in liquid nitrogen, Apimondia, Prague, 1975, pp. 311–314. [14] A. Oleksa, I. Chybicki, A. Tofilski, J. Burczyk, Nuclear and mitochondrial patterns of introgression into native dark bees (Apis mellifera) in Poland, J. Apic. Res. 50 (2011) 129–160. [15] M. Osanai, B. Baccetti, Two-step acquisition of motility by insect spermatozoa, Experientia 49 (1993) 593–595. [16] S. Pitnick, M.F. Wolfner, S.S. Suarez, Ejaculate-female and sperm-female interactions, in: T.R. Birkhead, D.J. Hosken, S. Pitnick (Eds.), Sperm Biology – An Evolutionary Perspective, Academic Press, Burlington, San Diego, Oxford, 2009, pp. 247–304. [17] F. Ruttner, Zuchttechnik und Zuchtauslese bei der Biene, Ehrenworth, Munich, 1996. [18] F. Ruttner, G. Koeniger, Filling of spermatheca of honeybee queen – active migration or passive transport of spermatozoa, Zeitschrift für vergleichende Physiologie 72 (1971) 411–422. [19] S.S. Schneider, L.J. Leamy, L.A. Lewis, G. DeGrandi-Hoffman, The influence of hybridization between African and European honeybees, Apis mellifera, on asymmetries in wing size and shape, Evolution 57 (2003) 2350–2364. [20] SPSS, IBM SPSS Statistics 19 core system user’s guide, SPSS, 2010. [21] M.A. Taylor, E. Guzmán-Novoa, N. Morfin, M.M. Buhr, Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios, Theriogenology 72 (2009) 149– 159. [22] D. vanEngelsdorp, J.D. Evans, C. Saegerman, C. Mullin, E. Haubruge, et al., Colony collapse disorder: a descriptive study, PLoS ONE 4 (2009) e6481. http:// dx.doi.org/10.1371/journal.pone.0006481. [23] L.R. Verma, An ionic basis for a possible mechanism of sperm survival in the spermatheca of the queen honey bee (Apis mellifera L.), Comp. Biochem. Physiol. A Comp. Physiol. 44 (1972) 1345–1351. [24] L.R. Verma, Osmotic analysis of honeybee (Apis mellifera L.) semen and haemolymph, Am. Bee J. 113 (1973) 412. [25] J. Wegener, K. Bienefeld, Toxicity of cryoprotectants to honey bee semen and queens, Theriogenology 77 (2012) 600–607. [26] J. Wegener, T. May, G. Kamp, K. Bienefeld, New methods and media for the centrifugation of honey bee (Hymenoptera: Apidae) drone semen, J. Econ. Entomol. 107 (2014) 47–53. [27] J. Wegener, T. May, U. Knollmann, G. Kamp, K. Bienefeld, In vivo validation of in vitro quality tests for cryopreserved honeybee semen, Cryobiology 65 (2012) 126–131.
