Female Pygmy Squid Cryptically Favour Small Males and Fast Copulation as Observed by Removal of Spermatangia

Females can express mate (or fertilisation) preferences after copulation. In the Japanese pygmy squid, Idiosepius paradoxus, in which males do not show any conspicuous pre-copulatory displays, the females remove the spermatangia attached to their bodies after copulation. In this study, we observed pre- and post-copulatory behaviours and analysed which variables associated with copulation were correlated with spermatangia removal. When females mated with larger males or copulation lasted longer female squid elongated their buccal mass after copulation and removed more spermatangia. We also investigated the effects of spermatangia removal on the retained spermatangia to predict whether cryptic female choice (CFC) influenced fertilisation success. Spermatangia removal by females had a stronger effect on the number of spermatangia retained than did the number of spermatangia ejaculated by males. These results suggest that spermatangia removal after copulation by buccal mass elongation works as a CFC in Japanese pygmy squid, and females cryptically favoured small males and fast copulation.     

Spermatophoric reaction reappraised: Novel insights into the functioning of the loliginid spermatophore based on Doryteuthis plei (Mollusca: Cephalopoda)

During copulation, spermatophores produced by male coleoid cephalopods undergo the spermatophoric reaction, a complex process of evagination that culminates in the attachment of the spermatangium (everted spermatophore containing the sperm mass) on the female's body. To better understand this complicated phenomenon, the present study investigated the functional morphology of the spermatophore of the squid Doryteuthis plei applying in vitro analysis of the reaction, as well as light and electron microscopy investigation of spermatangia obtained either in vitro, or naturally attached on females. Hitherto unnoticed functional features of the loliginid spermatophore require a reappraisal of some important processes involved in the spermatophoric reaction. The most striking findings concern the attachment mechanism, which is not carried out solely by cement adhesive material, as previously believed, but rather by an autonomous, complex process performed by multiple structures during the spermatophoric reaction. During evagination, the ejaculatory apparatus provides anchorage on the targeted tissue, presumably due to the minute stellate particles present in the exposed spiral filament. Consequently, the ejaculatory apparatus maintains the attachment of the tip of the evaginating spermatophore until the cement body is extruded. Subsequently, the cement body passes through a complex structural rearrangement, which leads to the injection of both its viscid contents and pointed oral region onto the targeted tissue. The inner membrane at the oral region of the cement body contains numerous stellate particles attached at its inner side; eversion of this membrane exposes these sharp structures, which presumably adhere to the tissue and augment attachment. Several naturally attached spermatangia were found with their bases implanted at the deposition sites, and the possible mechanisms of perforation are discussed based on present evidence. The function of the complex squid spermatophore and its spermatophoric reaction is revisited in light of these findings. J. Morphol. 2012. © 2011 Wiley Periodicals, Inc.     

Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns

The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0 ± 11.9%) was significantly higher (p = 0.0006) than the mean of the control group (7.5 ± 4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6–38.0%) and the frequency of genetically normal/balanced gametes (34.3–62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R = 0.4524, p = 0.2604; AB: R = 0.5238, p = 0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure.     

Effects of male mating interval on spermatophore formation,transfer, and subsequent female receptivity and fecundity in the sorghum plant bug,Stenotus rubrovittatus

Males of the sorghum plant bug, Stenotus rubrovittatus (Matsumura) (Heteroptera: Miridae), transfer a spermatophore to females during copulation. After a 1‐day interval between the first and second copulation, males transferred both sperm and a spermatophore to females during the second copulation. However, when male mating interval was <1 h, they transferred sperm but no spermatophores to females during the second copulation. Therefore, the male mating interval probably produces two types of mated females, those with and those without a spermatophore. Mated females of S. rubrovittatus do not remate for at least 3 days after mating, even when courted, and lay more eggs than virgin females at the beginning of the oviposition period. The effects of spermatophores on female sexual receptivity and fecundity were examined using mated females with or without a spermatophore. Only one of the 40 (2.5%) mated females with a spermatophore remated, whereas 10 of the 26 (38.5%) without a spermatophore remated. Furthermore, mated females with a spermatophore laid more eggs than those without a spermatophore. These results suggest that spermatophores participate in reducing female sexual receptivity and enhancing female fecundity in S. rubrovittatus.     

Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm

Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8 h) on survival before freezing at 4 °C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates = 6), pooled and diluted with Tris-citrate extender. Pooled semen samples were subjected to either RC (−2.2 °C/min) or MC (−0.3 °C/min) from 37 °C to 4 °C in separate aliquots and further equilibrated at 4 °C for 8 h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8 h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4 °C, of motility and PMI was observed 5 and 6 h respectively in RC group while >8 h in MC group. Rate of decline (slope) in motility and viability was higher (P < 0.05) in RC overtime during equilibration at 4 °C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P < 0.05) in MC when equilibrated for 2–8 h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2–8 h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.     

Giant spermatozoa and a huge spermatheca: A case of coevolution of male and female reproductive organs in the ground louse Zorotypus impolitus (Insecta,Zoraptera)

The male and female genital apparatus of the recently discovered ground louse Zorotypus impolitus were examined using light and electron microscopy. The rounded testes and a large seminal vesicle are connected with a complex of four accessory glands by a long tapering ejaculatory duct. Two accessory glands have the same whitish coloration, whereas the third one is pale blue, and the elongated and cylindrical fourth one translucent. The sperm are the largest known in Hexapoda, 3 mm long and 3 μm wide, with a volume of ca. 21,000 μm³; the ratio between the diameter of the axoneme and the width of the main body of the sperm ranges between 1:10 and 1:13. The exceptional width of the spermatozoa is due to an extreme enlargement of the mitochondrial derivatives and accessory bodies. A single sperm is contained in a small globular spermatophore (100 μm). The highly unusual external transfer correlates with an atypical mating behavior. The male produces several to many spermatophores during the mating process. As in other zorapterans the ovaries are panoistic and the eggs bear two micropyles. An exceptionally large apical spermathecal receptacle is present; it is connected with the vagina by a long spermathecal duct, which varies structurally along its course. A correlation between the sperm size and the size of the spermatheca is likely. Ultrastructural features of different species support two strikingly different models of male and female reproductive apparatus in the small order Zoraptera. This is in stark contrast to the extreme uniformity of their external morphology. It is likely that sexual selection played a decisive role in the evolution of the reproductive system.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

Toxicity evaluation of parboiled rice effluent using sperm quality of zebrafish as bioindicator

This study aimed to assess the acute toxicity of raw and treated wastewater generated by the rice parboiling industry using zebrafish (Danio rerio) sperm quality as a bioindicator. Toxicity bioassays were conducted comparing physicochemical parameters of sperm quality for zebrafish at sublethal conditions (n = 150 fish, 50 per treatment). Acute toxicity was detected in all sperm quality parameters assessed for both raw and treated wastewater, when contrasted to the control (p < 0.05). For zebrafish exposed to raw effluent, negative correlations with parameters of sperm quality were observed for the concentration of iron, phosphorus and total suspended solids (p < 0.05). Salinity, the biochemical oxygen demand and the concentration of total suspended solids were negatively correlated with parameters of sperm quality for zebrafish exposed to treated effluent (p < 0.05). In comparison with the levels observed for the raw effluent, most physicochemical parameters of the treated effluent were reduced to levels within the limits required by the environmental legislation. Despite the physical and chemical parameters measured in the treated wastewater meeting environmental legislation thresholds, acute toxicity persisted. These results show that the sperm quality can be used as a bioindicator for wastewater toxicity and release of wastewater to surface water could affect the fertility of fishes.     

Impact of cryptic female choice on insemination success: Larger sized and longer copulating male squid ejaculate more,but females influence insemination success by removing spermatangia

In polyandrous mating systems, sperm competition and cryptic female choice (CFC) are well recognized as postcopulatory evolutionary forces. However, it remains challenging to separate CFC from sperm competition and to estimate how much CFC influences insemination success because those processes usually occur inside the female's body. The Japanese pygmy squid, Idiosepius paradoxus, is an ideal species in which to separate CFC from sperm competition because sperm transfer by the male and sperm displacement by the female can be observed directly at an external location on the female's body. Here, we counted the number of spermatangia transferred to, removed from, and remaining on the female body during single copulation episodes. We measured behavioral and morphological characteristics of the male, such as duration of copulation and body size. Although males with larger body size and longer copulation time were capable of transferring larger amounts of sperm, females preferentially eliminated sperm from males with larger body size and shorter copulation time by spermatangia removal; thus, CFC could attenuate sperm precedence by larger males, whereas it reinforces sperm precedence by males with longer copulation time. Genetic paternity analysis revealed that fertilisation success for each male was correlated with remaining sperm volume that is adjusted by females after copulation.     

Optimization of short-term storage of pikeperch semen: an applicable approach

Contamination of semen with urine and asynchronous maturation of males and females are main obstacles in artificial reproduction of pikeperch Sander lucioperca. The objective of this study was to overcome these obstacles using optimization of a procedure for short-term storage of pikeperch semen at 4 °C using two immobilizing media (IM): (a) IM1, 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂ ⋅ 2H₂O and 2.38 mM NaHCO₃, 343 mOsm/kg; and (b) IM2, 200 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂ ⋅ 2H₂O and 2.38 mM NaHCO₃, 381 mOsm/kg. Undiluted sperm was used as the control. At 6 h poststorage, there were no substantial changes in spermatozoa motility and velocity at 30 s postactivation in all groups. Over 48 h of storage, the highest spermatozoa motility and velocity were obtained in sperm diluted in IM2 compared to the other groups. IM2 could maintain a significantly higher ATP content of diluted sperm than IM1 and undiluted treatment for 2 days. Similarly, the highest values of eyeing and hatching rates were observed in sperm diluted in IM2 compared to sperm in the other studied groups. It can be concluded that the obtained result is a novel and applicable approach to maintain semen quality of pikeperch during short-term storage, suggesting IM2 as a promising medium for short-term storage. The present study also opens possibilities for ensuring a reliable source of semen as a convenient approach for increasing genetic diversity in hatcheries.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Motility and fertility of cryopreserved semen in Persian sturgeon,Acipenser persicus,stored for 30–60 min after thawing

We investigated the effect of storage times of frozen–thawed Persian sturgeon (Acipenser persicus) semen on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30, and 60 min at 4 °C post-thawing. Frozen thawed semen analyzed immediately after thawing had similar quality characteristics as fresh semen. For cryopreserved semen stored for 30 min after thawing the characteristics did not differ to fresh semen and cryopreserved semen. For cryopreserved semen stored for 60 min a significant decline in the parameters was observed. Fertilization and hatching rates were not affected by storage times of maximally 30 min of storage.     

Protective effect of taurine,glutathione and trehalose on the liquid storage of ram semen

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes for the decline in motility and fertility during storage—the other detrimental cause is low temperature on the destabilisation of sperm membrane structure. The aim of this study was to determine the effects of the addition of the anti-oxidants taurine and glutathione (GSH), and the membrane structure stabiliser, trehalose, on sperm viability during low temperature liquid storage. A total number of 36 ejaculates were collected using the artificial vagina from four Chios rams and nine replicates of the ejaculates were diluted with a Tris-based extender containing additives as the control. The sperm motility, percentage abnormal sperm, plasma membrane intact sperm and the hypo-osmotic swelling test (HOST) were determined during storage of semen at 5 °C for a period of 0, 6, 24 and 30 h of liquid storage, respectively. Trehalose at a level of 50 mM provided the best maintenance of motility at 6 and 30 h (P < 0.05), and gave the highest percentage (69.0 ± 2.0% and 64.6 ± 1.8%, respectively) of viable sperm at 24 and 30 h (P < 0.01). Trehalose treatment at a concentration of 50 mM also resulted in the highest percentage of membrane-intact sperm (53.7 ± 2.9%) after performing HOST at 30 h. The anti-oxidant treatments GSH 5–10 mM and taurine at 50 mM provided a significant improvement in sperm survival during the 6 h of liquid storage at 5 °C (P < 0.05). In conclusion, many aspects of sperm protection, e.g. sperm motility, viability and membrane stabilisation of the sperm cells during relative low temperature storage, are the key factors determining the preservation of sperm function. Future efforts toward improving function of ram sperm kept in low temperature storage should concentrate on anti-oxidant additives. The results of this study provide a new approach to the preservation of sperm from rams of the Chios and related breeds, and so contribute to the improvement of these breeds for the world sheep industry.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

Relationship between pregnancy,embryo development,and sperm deoxyribonucleic acid fragmentation dynamics

The way the dynamics of DNA fragmentation affects the growth of embryos in real time, and effectiveness of infertility treatment using the ICSI procedure were determined in 148 couples treated with the ICSI technique. The percentage of sperm with fragmented DNA (known as the DNA fragmentation index [DFI]) in semen samples was determined at 3, 6 and 12 h. Embryo culture was assessed continuously during 12 h of observation monitoring.Statistically significant difference was found in DFI at 12 h and outcome of treatment. For the remaining time intervals, no statistically significant differences were noted. An analysis of relationship between the DFI dynamics over time at individual measurements and achievement of pregnancy, confirmed a statistically significant relationship between the rate measured at 6–12 h of observations of DFI changes (DFI 12 h%/h), and achieving pregnancy. Correlation was observed between DFI (during 0, 3, 6 and 12 h), the growth rate in DFI, and time of embryo development. A statistically significant relationship was found between the rate from the start to the end of observations of the DFI, and outcome of treatment.Intensity level regarding fragmentation of sperm DNA and its growth rate affected the time of embryo development in the ICSI procedure. The most significant prognostic factor for achieving pregnancy was intensification of sperm DNA fragmentation after 12 h.     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Dogs with Leishmania chagasi infection have semen abnormalities that partially revert during 150 days of Allopurinol and Amphotericin B therapy

The goal of the present study was to characterize the semen quality of dogs naturally infected with Leishmania chagasi, and treated with Allopurinol and Amphotericin B. Eight naturally infected and eight non-infected dogs were selected. Following semen collection, progressive motility, vigor, concentration and sperm morphology were evaluated. The seminal patterns in the treated animals were evaluated at the beginning (d0) and at days 30 (d30), 60 (d60) and 150 (d150) of treatment. The progressive motility at d0 (35.7 ± 22.3%) was less than that of the control group (77.8 ± 7.1%) (P < 0.05). The vigor was similar to the control group throughout the treatment (P > 0.05). The number of sperm/mL, sperm/ejaculate and sperm/kg of body weight was similar among groups (P > 0.05). The percentages of normal spermatozoa of infected and treated animals were similar throughout the treatment and to the control group (69.1 ± 8.7%) at d60 (37.5 ± 11.2%) and d150 (48.3 ± 10.8%) (P > 0.05), but smaller at d0 (22.7 ± 10.5%) and d30 (28.8 ± 15.9%) (P < 0.05). A greater percentage of acrosome damage was observed in the control group (3.1 ± 2.3%) compared to the d60 (0.1 ± 0.2%) (P < 0.05). The infected dogs had a greater percentage of principal piece defects at d60 (37.0 ± 6.3%) than the control group (16.8 ± 7.3%) (P < 0.05); and greater percentages of detached normal heads at d0 (28.7 ± 19.7%) and d30 (18.5 ± 18.5%) than the control group (0.4 ± 0.5%) (P < 0.05). This reduction in semen quality of the infected animals is suggestive of an epididymal dysfunction. Due to this poor semen quality, caution is recommended when using infected male dogs for reproductive purposes.     

Different sperm number in the spermatophores of Orchesella villosa (Geoffroy) (Entomobryidae) and Allacma fusca (L.) (Sminthuridae)

Members of the basal hexapod group Collembola perform indirect sperm transfer; males deposit spermatophores on the soil and these are then picked up by females for insemination. Orchesella villosa exhibits a dissociated mating behavior, while Allacma fusca has either associated mating behavior or, more commonly, produces spermatophores without pairing. Sperm ultrastructure in the two species has been studied by TEM and SEM observations to estimate the volume of these cells. The sperm number into each spermatophore has been determined by counting the fluorescent nuclei after Hoechst staining of sperm droplets squash preparations. Sperm droplets at the apex of spermatophores contain numerous spermatozoa of typical appearance, wound up and with a long anterior appendage (peduncle). Sperm droplets of Orchesella villosa are smaller (about 10 times) than in A. fusca, however they contain substantially more sperm cells (about three times). These differences could be explained as follows: (a) sperm of O. villosa are shorter than those of A. fusca (58 and 107 μm, respectively) and thus the volume of a single sperm of O. villosa is about 10 times smaller than in A. fusca; (b) considering the volume of sperm droplets and of sperm cells, the estimated number of sperm cells would be higher in A. fusca than in O. villosa; and (c) the conspicuous reduction of the sperm cells observed in A. fusca is likely linked to the aberrant meiosis present in this species, as well as in all Symphypleona studied so far, that causes a loss of 50% of their sperm.     

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.     

Mating effort function of the spermatophore in the bushcricket Poecilimon veluchianus (Orthoptera, Phanero[pteridae): support from a comparison of the mating behaviour of two subspecies.

To analyse spcrmatophore function, various aspects of the mating behaviour (e.g. spermatophore mass, duration of sperm transfer, mating frequency) were compared in two subspecies of the bushcricket P. veluchianus. Body mass was significantly different in both subspecies and had a strong effect on spermatophore mass, resulting in a large difference in absolute and relative (percentage of male body mass) spermatophore mass in both subspecies. After copulation the small P. v. minor spermatophores were consumed much faster by the female than the larger ones of P. v. veluchianus. The time necessary for sperm transfer from the spermatophore to the female spermatheca was much shorter in the subspecies with small spermatophores than in that with large spermatophores, and in both subspecies similar to the time required to consume the spermatophores. Mating frequency varied also between the subspecies and was murh higher in P. v. minor than in P. v. veluchianus. Differences in body mass between the two subspecies therefore resulted in changes in several aspects of mating behaviour. However, despite large differences in the mass of the spermatophore, its main function seems to be to ensure sperm transfer. This can be concluded from the similarity between the duration of sperm transfer and the time period necessary for spermatophore consumption in each of the subspecies. The spermatophore is thus considered to be male mating effort.