The effects of nonpenetrating cryoprotectants added to TEST-yolk-glycerol extender on the post-thaw motility of ram spermatozoa

The effects of adding five different concentrations of 17 polymeric compounds to TEST-yolk-glycerol extender on ram spermatozoa survival was studied. These were Aquacide (I, II, and III); dextran (0.8-1.6, 1.9, 15-20, 70, and 200-300 kDa); three types of Dri-Sweet; hydroxyethyl starch; methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and Supercol 912. All the compounds tested except the Dri-Sweet compounds and hydroxyethyl starch significantly (P less than 0.05) decreased percentages of motile cells in unfrozen samples. The use of dextran (0.8-1.6 kDa; hydrolyzed dextran separated by ethanol) and Aquacide II significantly (P less than 0.05) increased post-thaw motility of spermatozoa frozen in pellets. Dextran (15-20 kDa), dextran (0.8-1.6 kDa), Aquacide II, and hydroxyethyl starch significantly (P less than 0.05) increased the percentages of post-thaw motility of ram spermatozoa frozen in the presence of glycerol and egg yolk.     

Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina)

We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes–epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide – DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing–thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.     

Effects of different concentration and combinations of cryoprotectants on sperm quality,functional integrity in three Indian horse breeds

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4–6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.     

Improvement of the post-thaw qualities of Okinawan native Agu pig sperm frozen in an extender supplemented with antiapoptotic PTD-FNK protein

The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native Agu pig. The objective was to determine whether an artificial anticell death protein (PTD-FNK protein) was capable of improving the quality of cryopreserved Agu sperm. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 300, or 400 nm PTD-FNK protein was thawed, and mitochondrial integrity and other sperm characteristics were evaluated. Treatment with 300 nm PTD-FNK protein had the most beneficial effect (P < 0.05) on mitochondrial integrity (45-59%) and sperm motility (56-67%) after freezing-thawing. In particular, the proportion of post-thaw sperm with activated caspase-9 and -3 but not caspase-8 was markedly reduced among sperm frozen in the presence of PTD-FNK protein (P < 0.05), implying protection against apoptotic-cell death in response to mitochondrial damage. There were high levels of intracellular ATP (9.4-10.5 nmol/10⁸ sperm) in post-thaw sperm treated with PTD-FNK protein, and the inhibitory effect of PTD-FNK protein on activation of caspases influenced the increase in the number of sperm with intact DNA (36-53%; P < 0.05). Furthermore, the addition of PTD-FNK protein to the freezing extender strongly preserved the ability of the sperm to penetrate to mature oocytes in all individuals (60-80%; P < 0.05). In conclusion, treatment with PTD-FNK protein in the freezing extender effectively improved post-thaw qualities of fragile Agu sperm through prevention of mitochondrial dysfunction leading to apoptotic-cell death during cryopreservation.     

Surface changes associated with ram sperm cryopreservation revealed by counter-current distribution in an aqueous two-phase system effect of different cryoprotectants

Ram sperm was frozen in the presence of the most commonly used cryoprotectants. After thawing, the overall cell surface changes provoked by freezing were assessed by centrifugal counter-current distribution (CCCD). In addition, cell membrane integrity (viability) of all the treated sperm was estimated by fluorescent staining. Fresh and refrigerated sperm were used as controls. Our results show no improvement of the cooling-induced cell surface damage by freezing in the presence of bovine seminal plasma, proline, glycine-betaine and phosphatidylcholine. Better results were obtained with vitamin E and cholesterol. However, the best protective effects were found by employing seroalbumin and lactalbumin. Furthermore, freezing in the presence of bovine lactalbumin resulted in a good maintenance of the cellular viability and of the CCCD heterogeneity in respect to fresh cells.     

The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm

Adding cryopreservation media with reduced glutathione (GSH) has previously been shown to maintain the motility, membrane integrity and fertilizing ability of frozen-thawed boar sperm, although the effects of GSH on good (GFE) and poor freezability (PFE) ejaculates rely upon the intrinsic ejaculate freezability. The resilience to withstand freeze-thawing procedures has previously been related to the existence of a specific distribution of motile sperm subpopulations, which differs between GFE and PFE. Thus, the main aim of this study was to determine whether the addition of GSH to freezing media has any impact on the distribution of motile sperm subpopulations in GFE and PFE. With this purpose, 18 GFE and 13 PFE were cryopreserved with or without 2 mM GSH. Sperm quality and motile subpopulations were evaluated at 30 min and 4 h post-thawing. Three subpopulations were identified and the percentages of spermatozoa belonging to the fastest and most linear subpopulation, which was referred as ‘SP1’, decreased over post-thawing time. Good freezability ejaculates that were cryopreserved in the presence of 2 mM exhibited a significantly higher percentage of spermatozoa belonging to SP1 than the other combinations of treatment and freezability both at 30 min (mean ± SEM: GFE-C: 16.6 ± 0.4; GFE-GSH 27.7 ± 0.6) and 4 h post-thawing (GFE-C: 7.8 ± 0.2 vs. GFE-GSH: 16.7 ± 0.4). In conclusion, the positive effect of GSH on the motility of frozen-thawed sperm is related to a specific sperm subpopulation (SP1), which could coincide with the fertile sperm one.     

Cryopreservation of the endangered mahseer (Tor khudree) spermatozoa: I. Effect of extender composition, cryoprotectants, dilution ratio, and storage period on post-thaw viability

Several in situ and ex situ conservation strategies have been suggested for the revival of stocks of Tor khudree (Sykes), a threatened species. Cryopreservation of spermatozoa is crucial for the conservation of stocks of endangered species so that sustainable production can be ensured. Among the different extenders, modified fish Ringer (E1) was found to be the best for cryopreservation of T. khudree spermatozoa. Extender E2 appeared the next best. Extenders based on chicken egg yolk and milk powder were found to be unsuitable for the cryopreservation of T. khudree spermatozoa. Among the cryoprotectants, dimethyl sulfoxide provided maximum protection to spermatozoa during freezing and thawing. Propylene glycol and methanol were found to be less effective. Of the four spermatozoa dilutions, 1:10, 1:15, and 1:20 showed better motility rates than 1:5. At the former dilution ratios, the motility rates which were more than 95% prior to freezing were reduced to 80-81 and 43-67%, 10 and 70 days after cryopreservation, respectively. The motility duration did not differ much with increasing storage period at all the dilution ratios. Motility rates generally decreased with an increase in frozen storage. When spermatozoa were thawed and stored at 25 degrees C for varying periods, motility percentage, and duration decreased gradually as the storage period increased; spermatozoa stored up to 40 min after thawing retained 55% motility and were motile up to 77s; these values declined further leading to the complete cessation of motility 70 min after storage. The importance of extender-cryoprotectant mixture, milt dilution, and storage period in developing a protocol for T. khudree spermatozoa cryopreservation is discussed.     

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p < 0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p < 0.05).     

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Equine spermatozoa stored in the epididymis for up to 96 h at 4 °C can be successfully cryopreserved and maintain their fertilization capacity

After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4 °C up to 96 h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing–thawing on viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate (ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96 h post castration. The average volume (720 ± 159 μL) and the concentration (6.5 ± 0.4 × 10⁹ spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage. Sperm viability after refrigeration at 4 °C for up to72 h was similar (P < 0.01). The effect of sperm dilution in the freezing media showed similar values up to 48 h, while viability was preserved up to 72 h (P < 0.01). Cryopreserved spermatozoa show similar viability between different storage times. Chromatin condensation was not affected by storage time; however, incubation for 30 min in freezing medium and freezing–thawing process induced an increase in the chromatin decondensation. ROS generation was not affected by storage up to 96 h. Epididymal storage did not affect sperm protein tyrosine phosphorylation patterns; although the pattern of phosphorylation changed to strong staining of the equatorial segment when the sperm where capacitated in sperm–TALP. Finally, successful and similar pronuclear formation (analyzed by ICSI) and in vitro penetration (evaluated with bovine zone free oocyte) was observed using cryopreserved sperm obtained from prolong epididymal storage at 4 °C. In conclusion, cryopreservation of epididymal stallion sperm stored for up to 72 h in the epididymis at 4 °C, maintain both viability and ability to fertilize in vitro.     

Postthaw survival of rhesus macaque sperm: variation in the response of individual males to different freezing protocols

This study was designed to assess the effects of various cryopreservation protocols on the postthaw survival of rhesus macaque sperm. Multiple ejaculates of five mature males were collected and frozen by each of four different methods. Cryopreservation significantly reduced motility in all samples, regardless of the method, and the response of different ejaculates of the same male to each method proved to be consistent. The freezing method was a significant factor in determining the immediate postthaw motility, although this effect disappeared after 4 hr. Moreover, there was a significant interaction between individual males and freezing method suggesting that developing a single freezing protocol that is universally suitable may be difficult.     

Boar spermatozoa and prostaglandin F2alpha. Quality of boar sperm after the addition of prostaglandin F2alpha to the short-term extender over cooling time

Prostaglandin F(2alpha) (PGF(2alpha)) has been used to improve reproductive performance in swine. The goal of the present work was to determine how the addition of PGF(2alpha) affects boar sperm quality. Eleven different treatments were evaluated: eight with only PGF(2alpha) (0.625, 1.25, 2.50, 5, 10, 12.50, 25 and 50mg PGF(2alpha)/100ml) and three binary treatments (0.625mg PGF(2alpha)/100ml+200mug/ml hyaluronic acid (HA), 1.25mg PGF(2alpha)/100ml+200mug/ml HA, 0.625mg PGF(2alpha)/100ml+7.5muM caffeine (Caf)). All these substances were added to 16 ejaculates from 16 healthy and sexually mature boars (n=16), and each ejaculate was considered as a replicate. Our study also assessed the effects of these 11 treatments over different periods of preservation. Sperm quality was tested immediately after the addition of treatments (time 0), and after 1, 3, 6 and 10 days of cooling at 15 degrees C. To evaluate sperm quality, five parameters were analysed: (1) sperm viability, acrosome and mitochondrial sheath integrity (using a multiple fluorochrome-staining test), (2) sperm motility, (3) sperm morphology and (4) agglutination (using a computer assisted system) and (5) osmotic resistance (using the ORT). Parametric (analysis of variance for repeated measures) and non-parametric tests (Friedman test) were used as statistical analyses. Treatments with PGF(2alpha) concentrations higher than 12.5mg/100ml were cytotoxic while the others did not damage boar spermatozoa. Thus, the other treatments may be used to produce profitable effects without adverse effects. Moreover, the addition of PGF(2alpha) at 5mg/100ml to sperm diluted in BTS may maintain sperm viability and motility better after 6 days of cooling, because significant differences were observed (P<0.05) compared with control at the same time.     

Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. II: Effect of the addition of saccharides to freezing medium on sperm function

Saccharides have bioprotective properties, with a high capacity to preserve biological proteins and membranes during sperm cryopreservation. The aim of this study was to evaluate how replacing the lactose of cryopreservation media by sucrose (SUC) or trehalose (TRE) at concentrations of 0.2 M (SUC-1 and TRE-1) and 0.25 M (SUC-2 and TRE-2) affects frozen/thawed pig spermatozoa. The media used were composed of medium A (saccharide/egg yolk) and B (saccharide/egg yolk/glycerol), their osmolality being determined prior to freezing. Cell viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), lipid peroxidation, thiol group oxidation, total reactive oxygen species (ROS), peroxynitrite and superoxide anion (O₂^●-) were determined through flow cytometry; total motility (TM), progressive motility (PM) and kinetic parameters motility were determined immediately after thawing (T0) and again 30 (T30) and 60 (T60) minutes later. The SUC-2 and TRE-2 groups maintained viability significantly and presented fewer lipid membrane disorders, respectively, both with a significant increase in MMP. The production of O₂^●- and peroxynitrite was lower in the TRE-2 groups compared to the control (P < 0.05). Total motility at T0 was greater in the TRE-2 group (P < 0.05). Sperm kinetics was not affected by the treatment. The use of saccharides SUC and TRE at a concentration of 0.25 M improves sperm quality, so that both non-penetrating cryoprotectants can be utilized in pig sperm freezing media.     

Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig

The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 °C for up to 48 h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Cζ (PLCζ) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCζ in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18 h at 24 °C (78%, 62%, and 35%, respectively) and for 48 h at 14 °C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCζ in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.     

Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm

Two experiments were conducted to determine the effects of glycerol concentration and Equex STM® paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 °C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl®, cooled to 4 °C, stored at that temperature for 18-24 h, and further diluted with Biladyl® without or with Equex STM® paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 ± 4.6%; 3%: 20.5 ± 5.9% and 4%: 18.5 ± 6.6%; P > 0.05) or 3h post thaw (< 5% for all groups; P > 0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 ± 1.6%), 3% (81.3 ± 2.0%) and 4% glycerol (84.8 ± 2.0%; P > 0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 ± 3.8%) and 4% (77.2 ± 4.1%) than 2% glycerol (66.9 ± 2.7%; P < 0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM® (Equex®: 21.5 ± 3.5%; control: 14.4 ± 2.1%; P < 0.05) but was similar at 3h post-thaw (P > 0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM® paste immediately (control: 89.6 ± 1.2%; Equex®: 91.1 ± 1.4%; P > 0.05) and 3 h post-thaw (control: 69.3 ± 3.7%; Equex®: 59.9 ± 5.8%; P > 0.05). Sperm cryopreserved in medium containing 3-4% glycerol and 1% Equex STM® retained the best motility and acrosome integrity, even after liquid storage for 18-24 h at 4 °C prior to cryopreservation.     

Effect of the addition of beta-mercaptoethanol to a thawing solution supplemented with caffeine on the function of frozen-thawed boar sperm and on the fertility of sows after artificial insemination

We have reported that artificial insemination (AI) with frozen-thawed boar semen supplemented with caffeine increased the number of uterine sperm by inhibiting the migration of polymorphonuclear leukocytes (PMNs) into the uterine lumen, thereby improving the fertility of gilts and sows. The objective of the present study was to examine the effects of the addition of the antioxidant beta-mercaptoethanol (bME) and caffeine to the thawing solution on the function of frozen-thawed sperm, on the phagocytic activity of PMNs for sperm, and on the fertility of sows after AI. When frozen-thawed sperm were cultured in the presence of 25 or 50 μm bME, sperm capacitation and spontaneous acrosome reactions were inhibited (P < 0.01). There was no effect of bME on phagocytic activity of PMNs for sperm in vitro. When hormonally treated (400 IU of equine chorionic gonadotropin + 200 IU of human chorionic gonadotropin) weaned sows experienced a single intrauterine insemination with frozen-thawed sperm (25 × 10⁸ sperm per 50 ml dose) 40 h after subsequent hCG administration, pregnancy and farrowing rates were unaffected by the addition of 50 μm bME (pregnancy rate, 20 vs 21% in controls; farrowing rate, 20 vs 21%; n = 15 and 14, respectively). However, litter size tended to be higher than in the presence of 50 μm bME compared to its absence (10.0 ± 1.0 vs 5.7 ± 1.5, respectively; P < 0.07). Thus, the addition of bME to the thawing solution containing caffeine could be of benefit for improving the function of frozen-thawed sperm without influencing the phagocytic activity of PMNs for sperm. Although there were no statistically significant effects of bME on pregnancy or farrowing rates, the litter size tended to be higher in the sows subjected to a fixed-time single AI treatment with synchronized ovulation.     

Field evaluation of the response of Aedes albopictus (Stegomyia albopicta) to three oviposition attractants and different ovitrap placements using black and clear autocidal ovitraps in a rural area of Same,Timor‐Leste

Known oviposition attractants or stimulants were compared, singly and in combination, using inexpensive autocidal ovitraps designed to trap emerging adults, in a rural area of Timor‐Leste during the dry season. In this area, the dengue vector Aedes albopictus (Stegomyia albopicta) Skuse (Diptera: Culicidae) was abundant, but Aedes aegypti (Stegomyia aegypti) L. was not detected. The attractants were: (a) a compound found in Aedes eggs (dodecanoic acid); (b) components of nitrogen, phosphorous and potassium‐based (NPK) fertilizer, and (c) infusions of discarded cigarette butts. A solution of ammonium phosphate and potassium nitrate was significantly more attractive to gravid Ae. albopictus than water only. Dodecanoic acid and cigarette butt infusions were not significantly more attractive than the control; however, they attracted various other Diptera and many non‐culicid larvae developed in ovitraps in which these substances were used; thus, the presence of eggs or larvae of other species may have deterred Aedes oviposition. Significantly more Aedes eggs were found in ovitraps under vegetation than in ovitraps placed inside houses or against external walls. Clear‐sided ovitraps in which black mesh was placed over a black ring floating on the water surface collected significantly fewer eggs than black ovitraps with identically placed mesh and rings.