Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

Effect of bioactive peptide on ram semen cryopreservation

This present study investigated the effect of bioactive peptide (BAPT) (BAPT) on the quality of ram semen during cryopreservation. Ram ejaculates were extended with Tris buffer supplemented with no antioxidants (as control group), 20 μg/mL BAPT (as BAPT20 group), 40 μg/mL BAPT (as BAPT40 group) and 60 μg/mL BAPT (as BAPT60 group). After cryopreservation, sperm quality including motility, vitality, the percentage of hypoosmotic swelling test (HOST)-positive spermatozoa and the percentage of intact acrosomes was assessed. Furthermore, the malondialdehyde (MDA) in seminal plasma and spermatozoa were analyzed, followed by the measurement of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) levels in seminal plasma. After in vitro fertilization, the embryonic cleavage rates and development rates of different groups were analyzed to compare the developmental abilities of spermatozoa. The results showed that the post-thaw sperm motility was significantly higher in the BAPT60 group compared to those in the BAPT20, BAPT40 and control groups (P < 0.05). The percentage of live sperms significantly increased from 48.12 ± 2.35% for the BAPT20 group, 55.43 ± 2.16% for the BAPT40 group to 57.53 ± 3.15% for the BAPT60 group. The percentage of HOST-positive spermatozoa was significantly higher in the BAPT60 group than those in BAPT20, BAPT40 and control groups (P < 0.05). The MDA levels in seminal plasma and spermatozoa were significantly reduced with BAPT supplement (P < 0.05). Additionally, the SOD, CAT and GSH-Px levels in the BAPT experimental groups were significantly higher than those of the control group, which further indicated that BAPT significantly inhibit the reactive oxygen species (ROS) production during the cryopreservation of ram semen. Furthermore, the embryonic cleavage rates and development rates of the BAPT40 and BAPT60 groups were significantly increased in comparison with the BAPT20 and control groups (P < 0.05).In conclusion, BAPT improved the ram sperm quality via inhibiting the ROS production during cryopreservation, and could be applied as a promising supplement for ram semen cryopreservation.     

In vivo validation of in vitro quality tests for cryopreserved honey bee semen

Development of cryopreservation protocols for honey bee semen is hampered by the lack of validated laboratory tests that allow the prediction of in vivo performance of frozen-thawed semen. Here we analyzed correlations between seven in vitro tests and indicators of semen performance after insemination. These tests included measures of motility, cell conformation, and membrane permeability before and after exposure to physiochemical stress. We show that the proposed protocol for motility measurement yields results that correlate well with the number of sperm reaching the storage organ of queens (correlation coefficient ρ=0.67) and the proportion of viable eggs in inseminated queens (ρ=0.48). The conventional live/dead assay of membrane permeability by dual fluorescent staining and a new test based on the leakage of the glycolytic enzyme glucose-phosphate-isomerase (GPI) from damaged cells were also correlated to the number of sperm reaching the spermatheca (ρ=0.54 and -0.61, respectively). We conclude that motility, live/dead-staining and the assay for GPI-leakage are valuable tools for the improvement of cryopreservation of honey bee semen.     

Semen cryopreservation of yellow croaker <Emphasis Type="Italic">Larimichthys polyactis</Emphasis>

The effects of various extenders and cryoprotectants on movable spermatozoa ratio (MSR), spermatozoa velocity (SV) and duration of spermatozoa motility (DSM) of post-thawed spermatozoa were examined. The MSR, SV and DSM of post-thawed sperm in artificial seminal plasma (ASP) extender were higher than those in marine fish Ringer’s solution (MFRS) extender (P < 0.01) and was not significantly different from that of fresh sperm. No significant differences were observed in the motility parameters between fresh spermatozoa and frozen-thawed spermatozoa cryopreserved with ASP extender supplement 10% EG (ethylene glycol) cryoprotectant. Using the above method, yellow croaker semen was cryopreserved with extender ASP and 10% EG. As a result, at the spermatozoa/egg ratio of 100,000:1, the fertilization rate and hatching rate of the frozen-thawed spermatozoa cryopreserved for 1 week or 1 year in liquid nitrogen (45.7 ± 3.2% and 27.2 ± 5.0% or 37.5 ± 4.4% and 27.2 ± 5.0%) were similar to that of fresh spermatozoa (51.0 ± 3.1% and 36.7 ± 2.2%). There was a small alternation of shape in cryopreserved spermatozoa compared with fresh spermatozoa. In conclusion, the optimal conditions for yellow croaker semen cryopreservation are ASP extender supplement 10% EG cryoprotectant. This is the first report on semen cryopreservation of yellow croaker Larimichthys polyactis.     

Ginger and echinacea extracts improve the quality and fertility potential of frozen-thawed ram epididymal spermatozoa

The current study examined the impact of the supplementation of ginger and echinacea extract, as natural antioxidant agents, in freezing extender on the quality and fertility potential of ram epididymal spermatozoa after cryopreservation. Epididymal spermatozoa isolated from Forty testicles, obtained from 20 rams, with motility >80% and total morphological abnormalities <10% were pooled, divided into 7 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing ginger and echinacea extracts (5, 10 and 20 mg/l). The control diluent comprised of only extender and lacked any antioxidant agent. For the determination of sperm quality, frozen straws were thawed after 7–10 days, and then the sperm characteristics were assessed. The supplementation of ginger at a concentration of 10 mg/l, as well as the addition of 10 and 20 mg/l echinacea extract significantly improved total motility and velocity parameters. The status of acrosome integrity and lipid peroxidation significantly improved in spermatozoa when supplemented with 10 mg/l ginger and 20 mg/l echinacea extract. Also, 5 mg/l ginger extract and 20 mg/l echinacea extract significantly improved mitochondrial activity. The highest ratio of the dispersion of sperm chromatin was observed in spermatozoa treated with 10 mg/l ginger extract. The cleavage rate was markedly higher in matured oocytes that were fertilized with frozen spermatozoa treated with 20 mg/l ginger extract and 10 mg/l echinacea. The application of ginger and echinacea extract resulted in improvement in the quality and fertility of frozen-thawed spermatozoa. However, future studies are wanted to elucidate how the active components in these extracts prevent cryo-damages in spermatozoa.     

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 °C over 1.5 h (−0.16 °C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration–equilibration, after freezing and thawing, and 2 h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.     

Comparative reproduction of <Emphasis Type="Italic">Varroa destructor</Emphasis> in different types of Russian and Italian honey bee combs

Earlier studies showed that Russian honey bees support slow growth of varroa mite population. We studied whether or not comb type influenced varroa reproduction in both Russian and Italian honey bees, and whether Russian bees produced comb which inhibited varroa reproduction. The major differences found in this study concerned honey bee type. Overall, the Russian honey bees had lower (2.44 ± 0.18%) levels of varroa infestation than Italian honey bees (7.20 ± 0.60%). This decreased infestation resulted in part from a reduced number of viable female offspring per foundress in the Russian (0.85 ± 0.04 female) compared to the Italian (1.23 ± 0.04 females) honey bee colonies. In addition, there was an effect by the comb built by the Russian honey bee colonies that reduced varroa reproduction. When comparing combs having Russian or Italian colony origins, Russian honey bee colonies had more non-reproducing foundress mites and fewer viable female offspring in Russian honey bee comb. This difference did not occur in Italian colonies. The age of comb in this study had mixed effects. Older comb produced similar responses for six of the seven varroa infestation parameters measured. In colonies of Italian honey bees, the older comb (2001 dark) had fewer (1.13 ± 0.07 females) viable female offspring per foundress than were found in the 2002 new (1.21 ± 0.06 females) and 1980s new (1.36 ± 0.08 females) combs. This difference did not occur with Russian honey bee colonies where the number of viable female offspring was low in all three types of combs. This study suggests that honey bee type largely influences growth of varroa mite population in a colony.     

Survival of honey bee (Hymenoptera: Apidae) spermatozoa stored at above-freezing temperatures

The development of practical techniques for the storage of honey bee, Apis mellifera L., semen would significantly improve our ability to breed for desirable genotypes and maintain genetic diversity in populations. Artificial insemination of queens has been possible for some time, but the semen used is usually freshly collected, or held for < 1 wk at room temperature. I examined the limitations of spermatozoal survival at nonfrozen temperatures. Pooled, diluted semen was stored in sealed capillary tubes at room temperature (25 degrees C) or in a refrigerator set to 12 degrees C, for periods up to 1 yr. Survival of spermatozoa was assayed by a dual fluorescent staining technique using SYBR-14 and propidium iodide stains, which readily distinguishes live and dead cells. No significant loss of viable spermatozoa occurred within the first 6 wk. Between weeks 6 and 9, the percent live spermatozoa fell from 80 to 58%, and remained at that level until after 39 wk. By week 52, samples at room temperature, but not at 12 degrees C, fell to 18.9% live spermatozoa. Nonfrozen storage of honey bee semen has potential for short-term preservation of germplasm, however several factors need to be studied further to optimize survival rates.     

Effect of alpha-lipoic acid on oxidative stress,viability and motility in the common carp (Cyprinus carpio) spermatozoa after short-term storage and cryopreservation

As known for different metabolic functions, α-lipoic acid (ALA) has been tested for spermatozoa preservation of animals as well as of human, but not for fish spermatozoa. The present study determined the effects of ALA on short and long-term (cryopreservation) preservation of common carp (Cyprinus carpio) spermatozoa, for the first time. For that, spermatozoa were diluted in extenders containing 0 (control), 0.025, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM of ALA concentrations in both short-term preservation and cryopreservation. Spermatozoa motility parameters by computer-assisted semen analysis, viability, lipid peroxidation and catalase activity in spermatozoa were conducted in both 2nd and 120th hours of short-term storage and post-thaw samples. Higher percentages of total spermatozoa motility (80 ± 3) and viability (87 ± 3) were observed in 0.5 mM ALA group after 120 h of incubation. In post-thaw samples, higher percentages of these parameters were in 1 mM ALA group (74 ± 3 and 83 ± 2, respectively). Moreover, the results have shown that the addition of ALA until concentrations of 2 mM improved especially spermatozoa curvilinear velocity, maintained viability, and suppressed excessive lipid peroxidation during the preservations. In conclusion, the additions of 0.5 mM ALA for short-term preservation and 1 mM ALA for cryopreservation were the optimal concentrations, and shown the protective effects on common carp spermatozoa, when considering all measured parameters together.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

Sperm survival during short-term storage and after cryopreservation of semen from striped trumpeter (Latris lineata)

Methods of short-term storage and cryopreservation were examined for semen from striped trumpeter (Latris lineata). For fresh semen at 18 degrees C, the percentage of motile sperm declined rapidly from over 80% immediately after activation with sea water to less than 2% within 9 min after activation. The motility after activation of undiluted fresh sperm stored at 5 degrees C was maintained for two days and then declined markedly so that by the eighth day, sperm were mostly immotile after activation. The post-thawing motility was higher for sperm frozen with a non-activating diluent containing 2.84 M DMSO in saline (117 mM NaCl) than in an activating glycerol (2 M) medium in dilute sea water (300 mOsm). Post-thawing motility was higher for a dilution rate of 1:5 (semen:diluent) than 1:2 or 1:11 but was similar when frozen semen was thawed at 10 degrees, 20 degrees or 30 degrees C. For semen stored at a range of volumes as pellets frozen on dry ice (0.2 to 2.0 mL) or straws frozen in liquid nitrogen vapor (0.25 to 0.5 mL) and thawed in a waterbath at 20 degrees C, the post-thawing motilities were similar even though the patterns of cooling and thawing differed markedly between methods of freezing and sizes of pellets and straws.     

Detection of multiple viruses in queens of the honey bee Apis mellifera L

Individual honey bee Apis mellifera L. queens were examined for the presence of six honey bee viruses including acute bee paralysis virus, chronic bee paralysis virus, black queen cell virus, deformed wing virus, Kashmir bee virus, and sacbrood virus. All viruses, except ABPV, were detected in the samples. Among queens examined for virus infections, 93% had multiple virus infections. The detection of viruses in queens raises the possibility of a vertical transmission pathway wherein infected queens can pass virus through their eggs to their offspring.     

Sequential interval micro-droplet loading in closed hemi-straw carrier system: A convenient and efficient method for ultra-rapid cryopreservation in extreme oligozoospermia

Cryopreservation of human spermatozoa with low concentration while maintaining adequate post-thawing motility remains a major challenge for male fertility preservation. A convenient and efficient ultra-rapid freezing method for small amounts of human spermatozoa in a closed Hemi-Straw carrier system (CHS) was developed. Spermatozoa from 60 healthy men were involved in a parameter refining test and another 15 extreme oligozoospermic specimens were assigned to a verification test. A commercialized sperm freezing medium, Quinn's Advantage® Sperm Freeze medium (glycerol and sucrose as the cryoprotective agent) was used in the study. The results showed that the highest recovery rates would be obtained via the method of 2 μl single droplet sequential interval loading, by placing the straw at 1 cm above the liquid nitrogen (LN₂) surface for 60 s during freezing and 2 cm above the LN₂ for 2 min during thawing. This method was applied in cryopreservation for the normozoospermic specimens and compared with a conventional slow freezing method. The results were better than those in the control group in the total motility recovery rate (77.8 ± 11.2% vs 56.6 ± 11.9%, P < 0.01), progressive motility recovery rate (77.6 ± 13.2% vs 47.7 ± 14.6%, P < 0.01), 24 h survival index (60.9 ± 13.4% vs 42.1 ± 14.1%, P < 0.01) and the sperm DNA fragment index (4.2 ± 3.7% vs 5.8 ± 3.7%, P = 0.126). This method was applied to the oligozoospermic specimens. Motile spermatozoa could be found in 12 of 15 cases in the ultra-rapid freezing group, while only in 7 cases in control group. The results indicated that this freezing method was simple, convenient and bio-safe for cryopreservation of severe oligozoospermic specimens.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Effect of dilution rate on feline urethral sperm motility,viability, and DNA integrity

This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 10⁶/mL, 5 × 10⁶/mL, and 1 × 10⁶/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 10⁶/mL dilution rate, spermatozoa diluted to 1 × 10⁶ sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates.     

Cryogenic preservation of semen from the Aleutian Canada goose (Branta canadensis leucopareia)

Aleutian Canada geese (Branta canadensis leucopareia) were inseminated with frozen-thawed semen containing 6% or 7% dimethylsulfoxide (DMSO) resulting in 32 fertile eggs and 17 goslings; with 7% DMSO, 19 of 31 eggs were fertile. Beltsville Poultry Semen Extender (BPSE), adjusted to 270 ± 30 mOs and 7.5 ± 0.4 pH, was used to dilute semen samples and the DMSO before cryopreservation. About half of the live spermatozoa in the fresh semen (92.9 ± 2.5% live cells, laboratory studies; 87.3 ± 7.3%, insemination trials) survived the freeze-thaw process (46.7 ± 7.8%, laboratory; 33.3 ± 17.8%, insemination trials). Samples of frozen-thawed semen contained a greater percentage of bent spermatozoa (27.1 ± 8.4% of live cells) than fresh semen (14.4 ± 3.0% of live cells). Fecal- and urate-contaminated semen (a common problem when collecting goose semen) reduced the sperm motility score from 3.2 ± 0.6 to 2.7 ± 0.7 and number of live spermatozoa in frozen-thawed semen from 49 ± 9% to 24 ± 18%. Other variables examined that had less of an effect on semen quality included semen extenders, semen holding temperature, dilution and equilibration, relationship between hour of semen collection and level of semen contamination, and the relationship between season and sperm concentration.     

Testing the effect of paraquat exposure on genomic recombination rates in queens of the western honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

The rate of genomic recombination displays evolutionary plasticity and can even vary in response to environmental factors. The western honey bee (Apis mellifera L.) has an extremely high genomic recombination rate but the mechanistic basis for this genome-wide upregulation is not understood. Based on the hypothesis that meiotic recombination and DNA damage repair share common mechanisms in honey bees as in other organisms, we predicted that oxidative stress leads to an increase in recombination rate in honey bees. To test this prediction, we subjected honey bee queens to oxidative stress by paraquat injection and measured the rates of genomic recombination in select genome intervals of offspring produced before and after injection. The evaluation of 26 genome intervals in a total of over 1750 offspring of 11 queens by microsatellite genotyping revealed several significant effects but no overall evidence for a mechanistic link between oxidative stress and increased recombination was found. The results weaken the notion that DNA repair enzymes have a regulatory function in the high rate of meiotic recombination of honey bees, but they do not provide evidence against functional overlap between meiotic recombination and DNA damage repair in honey bees and more mechanistic studies are needed.     

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.     

A quantitative study of worker reproduction in queenright colonies of the Cape honey bee, Apis mellifera capensis

Reproduction by workers is rare in honey bee colonies that have an active queen. By not producing their own offspring and preventing other workers from producing theirs, workers are thought to increase their inclusive fitness due to their higher average relatedness towards queen-produced male offspring compared with worker-produced male offspring. But there is one exception. Workers of the Cape honey bee, Apis mellifera capensis, are able to produce diploid female offspring via thelytokous parthenogenesis and thus produce clones of themselves. As a result, worker reproduction and tolerance towards worker-produced offspring is expected to be more permissive than in arrhenotokous (sub)species where worker offspring are male. Here we quantify the extent to which A. m. capensis workers contribute to reproduction in queenright colonies using microsatellite analyses of pre-emergent brood. We show that workers produced 10.5% of workers and 0.48% of drones. Most of the workers' contribution towards the production of new workers coincided with the colonies producing new queens during reproductive swarming.