Sperm Types Research Articles

Mouse organ coefficient and abnormal sperm rate analysis with exposure to tap water and source water in Nanjing reach of Yangtze River

Organ coefficients (including kidney, testis, liver and spleen coefficient) and abnormal sperm rate were used in our study to reflect the exposure to the Yangzte River water. The concentrations of total dissolved metals and semi-volatile organic compounds in tap and source water were measured by ICP-OES and GC-MS, respectively. After mice were fed with purified water (CK), Nanjing tap water (NJT) and Nanjing source water (NJS) for 90 day, the individual and organs (including kidney, testis, liver and spleen) of each mouse were weighted. And abnormal sperm types (such as hook less, banana-like form, amorphous, folded and two tails) were determined by microscope. The results showed that significant differences of liver coefficient between experimental group (NJT, NJS) and control group (CK) were observed; furthermore liver coefficient is positive correlation with the concentrations of total dissolved metals. However, no significant differences of abnormal sperm rates between experimental group (NJT, NJS) and control group (CK) were noted. So liver coefficient might be more sensitive than other organ coefficients to reflect the exposure to tap water and source water, while abnormal sperm rate could not be used to reveal the exposure to them.

Comparative morphology of spermatozoa and reproductive systems of zorapteran species from different world regions (Insecta, Zoraptera)

The male and female reproductive apparatus of Zorotypus magnicaudelli (Malaysia), Zorotypus huxleyi (Ecuador) and Zorotypus weidneri (Brazil) were examined and documented in detail. The genital apparatus and sperm of the three species show only minor differences. The testes are larger in Z. magnicaudelli. Z. huxleyi lacks the helical appendage in the accessory glands. A long cuticular flagellum is present in Z. magnicaudelli and in the previously studied Zorotypus caudelli like in several other species, whereas it is absent in Z. weidneri, Z. huxleyi, Zorotypus hubbardi, Zorotypus impolitus and Zorotypus guineensis. Characteristic features of the very similar sperm are the presence of: a) two dense arches above the axoneme; b) a 9 + 9+2 axoneme with detached subtubules A and B of doublets 1 and 6; c) the axonemal end degenerating with enlarging accessory tubules; d) accessory tubules with 17 protofilaments; e) three accessory bodies beneath the axoneme; and f) two mitochondrial derivatives of equal shape. The first characteristic (a) is unknown outside of Zoraptera and possibly autapomorphic. The sperm structure differs distinctly in Z. impolitus and Z. hubbardi, which produce giant sperm and possess a huge spermatheca. The presence of the same sperm type in species either provided with a sclerotized coiled flagellum in males or lacking this structure indicates that a different organization of the genital apparatus does not necessarily affect the sperm structure. The flagellum and its pouch has probably evolved within Zoraptera, but it cannot be excluded that it is a groundplan feature and was reduced several times. The fossil evidence and our findings suggest that distinct modifications in the genital apparatus occurred before the fragmentation of the Gondwanan landmass in the middle Cretaceous.

Should older patients be offered varicocoele correction to improve their fertility?

We reviewed a large number of patients from 2000 to 2010 that underwent varicocoele correction using the retrograde sclerotization approach. Three hundred and seventy-six of them were included in the study, as they met the inclusion criteria. Mean age at the time of surgery was 32 years (SD: 6.5); 32% of them were 35 years and older. Patients were classified according to the clinical classification (GC) and Sarteschi's Doppler ultrasound classification (GS). The patients showed up at the clinic after an average time of 30 months of referred infertility related (SD: 28.54). Patients underwent pre-operative assessment with physical examination, sperm analysis and Doppler ultrasound, and again the same assessment at least 3 months after surgery. We evaluated the following parameters: sperm concentration (millions/mL, CONC), percentage motility (a+b, MOT) and the percentage of morphologically typical spermatozoa (MOR). Univariate and Multivariate analysis were performed. The research of Pearson's coefficients was performed to test the correlation between sperm parameters and age, SG, CG. Semen specimens were evaluated according to WHO 1999 criteria. Mean CONC varied from 34.5 to 47.0 millions/mL (+12.5; p < 0.001); MOT from 27.2 to 34.5% (+7.3%; p < 0.001); and MOR from 44.0 to 47.6% (+3.6%; p = 0.001). Univariable and multivariable analysis of variance related to age showed no significant difference of parameters improvement. Pearson's correlation coefficient for pre-operative and post-operative sperm MOT related to patients' age was respectively -0.11 (p < 0.001) and -0.18 (p = 0.04). No other significance was found. Usefulness of treating infertile patients affected by varicocoele is confirmed: varicocoele correction leads to significant sperm parameters improvement. There is no evidence of different improvement related to patients' age. The decline in sperm motility related to age of the patients seems to be only age-dependent: the usefulness of treating patients affected by varicocoele is not influenced by their age: treatment should also be offered to older patients.

Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program.

182 EFFECTS OF MITOCHONDRIAL ACTIVITY OF INJECTED SPERM ON EARLY DEVELOPMENT IN BOVINE INTRACYTOPLASMIC SPERM INJECTION-DERIVED EMBRYOS

Intracytoplasmic sperm injection (ICSI) has become the method of choice for bovine ovum pick-up and IVF. However, there are many difficulties with the ICSI technique to obtain viable fetuses. One of the major problems associated with this technique is our lack of knowledge concerning the status of the sperm mitochondria when injected into the oocyte and its effect on embryo development. First, we examined the mitochondrial activity of sperm that had been activated by culturing with methyl-β cyclodextrin (MBCD), in ICSI and in IVF. In vitro-matured oocytes and JC1-labelled sperm were used for the ICSI and IVF. The fluorescence intensity of injected/penetrated sperm mitochondria was measured using confocal laser scanning microscopy. Then, the relative membrane potential of the mitochondria was analysed by a ratiometric method. Second, the reactive oxygen species (ROS) production and capacitation status of the sperm exhibiting normal motility and of the sperm that had been activated by culturing with MBCD were analysed. The ROS levels produced by the sperm were estimated using the luminol assay. The chlortetracycline stain was used to evaluate capacitation status of the sperm. Third, the effect of ROS produced by these sperm types upon embryogenesis following ICSI and IVF was studied. Early developing embryos were examined with a stereomicroscope for cleavage and development to the blastocyst stage after 7 days of culture. Chromosome samples stained with Giemsa solution from the blastocysts were used to analyse the chromosomal integrity. Data were analysed by t-test for Experiments 1 and 2, and ANOVA with Fisher's PLSD test for Experiment 3. The mitochondrial activity immediately after ICSI was higher than at 3 h after insemination (immediately after sperm penetration) in IVF (P &lt; 0.05). The sperm exhibiting activation were capacitated and produced more ROS than the sperm exhibiting normal motility (P &lt; 0.05). The rates of cleaved embryo and blastocyst after ICSI with activated sperm were the same as that in ICSI with normal motility sperm and in IVF (cleaved rate: 66.7, 71.8, and 85.0%, respectively; blastocyst rate: 24.4, 23.3, and 32.0%, respectively). However, chromosomal integrity of blastocysts derived from ICSI with activated sperm was lower than that for ICSI with normal motility sperm or for IVF (23.1, 75.0, and 63.6%, respectively; P &lt; 0.01). In conclusion, capacitated, activated sperm induced chromosomal aberrations during early embryo development following ICSI. Conceivably, the selection of sperm exhibiting progressive motility, which is expected to be activated and to fertilize, would not always be better for early embryo development and fetal growth following ICSI due to the ROS derived from the sperm mitochondria. Injection of sperm exhibiting normal motility, or of mitochondria reduced activated sperm, could improve the quality of ICSI-derived embryos.

Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans

In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

First data on sperm heteromorphism in the mussels Crenomytilus grayanus and Mytilus trossulus (Bivalvia) from the Sea of Japan

Bivalve mollusks of the family Mytilidae inhabit almost all regions of the world ocean. Representatives of the genera Mytilus and Crenomytilus that are impor� tant objects of the mariculture and commercial catch� ing, dominate in coastal waters of the Sea of Japan. In the study of reproductive strategies of mytilids, their spermatozoa were described by a number of authors [3, 8, 12–14]. The data covered, mainly, peculiarities of morphology and ultrastructure of spermatozoa and traditionally were used for decision of questions in tax� onomy of this group of mollusks. Transmission elec� tron microscopy has shown that mytilids are charac� terized by a “primitive” type of the spermatozoon, which is usually found in invertebrate animals with external fertilization [10]. Until recent time, it has been considered that only one sperm type is inherent in each species of mytilids. However, we have revealed unusual cases of the heteromorphism in the sperm of Gray’s mussel Crenomytilus grayanus (Dunker, 1853) and the Pacific blue mussel Mytilus trossulus Gould, 1850. The goal of this study was detailed analysis of the heteromorphism phenomenon in male gametes of these two species. Mature gonads of mollusks, collected in the Vostok Bay (the Sea of Japan) in July–August 2011 were used for the study. Material was collected at the Vostok Marine Biological Station of the Institute of Marine Biology. Under laboratory conditions, gonads were removed and crashed into small pieces, which were fixed at 4°C in 2.5% glutaraldehyde in 0.2 M cacody� late buffer solution (pH 7.4) for 2–4 h. Then, the spec� imens were washed in 0.1 M cacodylate buffer solution for 15–20 min. The sperm suspension was placed by a pipette onto the surface of Thermanox cover slips (Cat. no. 72280), incubated until they sedimented (for 30 min), and then dehydrated in a series of accelerat� ing concentrations of ethanol, gradually bringing to a pure acetone. After that, the specimens were critical�

Integrated and independent evolution of heteromorphic sperm types

Sperm are a simple cell type with few components, yet they exhibit tremendous between-species morphological variation in those components thought to reflect selection in different fertilization environments. However, within a species, sperm components are expected to be selected to be functionally integrated for optimal fertilization of eggs. Here, we take advantage of within-species variation in sperm form and function to test whether sperm components are functionally and genetically integrated both within and between sperm morphologies using a quantitative genetics approach. Drosophila pseudoobscura males produce two sperm types with different functions but which positively interact together in the same fertilization environment; the long eusperm fertilizes eggs and the short parasperm appear to protect eusperm from a hostile female reproductive tract. Our analysis found that all sperm traits were heritable, but short sperm components exhibited evolvabilities 10 times that of long sperm components. Genetic correlations indicated functional integration within, but not between, sperm morphs. These results suggest that sperm, despite sharing a common developmental process, can become developmentally and functionally non-integrated, evolving into separate modules with the potential for rapid and independent responses to selection.

Cryptic choice of conspecific sperm controlled by the impact of ovarian fluid on sperm swimming behavior.

Despite evidence that variation in male–female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species’ ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species’ identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior.

Factors influencing intracytoplasmic sperm injection (ICSI) outcome in men with azoospermia

What's known on the subject? and What does the study add? The management of patients with non‐obstructive azoospermia (NOA) and some cases of obstructive azoospermia involves testicular sperm extraction (TESE or micro‐dissection TESE) combined with intracytoplasmic sperm injection (ICSI). Several studies have investigated the effect of the male age, the cause of azoospermia, testicular histopathology, the type of sperm used, and the use of pentoxyphilline, on the ICSI cycle outcome in men with azoospermia. The present study showed that none of these factors influenced the ICSI outcome in men with azoospermia, thus once sperm is found in an azoospermic male, no other male factor seems to influence the ICSI outcome. To our knowledge this is the first study to comment on the outcome of ICSI in men with NOA based on testicular histopathology. Objectives To access the effect of: male age, the cause of azoospermia (obstructive azoospermia vs non‐obstructive azoospermia [NOA]), testicular histopathology, the type of sperm used (fresh vs frozen‐thawed), and the use of pentoxyphilline on the intracytoplasmic sperm injection (ICSI) cycle outcome in men with azoospermia. To our knowledge this is the first study to comment on the outcome of ICSI in men with NOA based on testicular histopathology. Patients and Methods A retrospective analysis of 137 testicular sperm extraction‐ICSI cycles performed between 2001–2010, involving 103 men with azoospermia, with 26 couples having repeat cycles. Results Analysis of the results did not show any statistically significant differences in the fertilization, embryo cleavage, clinical pregnancy, live birth and miscarriage rates in relation to the male age, cuase of azoospermia, testicular histopathology, type of sperm used and the use of pentoxyphilline. Conclusion Once sperm is found in a man with azoospermia, no other male factor seems to influence the ICSI outcome.

Autonomous changes in the swimming direction of sperm in the gastropodStrombus luhuanus

The sperm of the gastropod Strombus luhuanus show dimorphism. The eusperm have a nucleus and fertilize the egg, whereas the other type of sperm, parasperm, are anucleate and are thought to assist fertilization. Here we report the autonomous changes in the swimming pattern of S. luhuanus eusperm. In artificial seawater, the eusperm collected from S. luhuanus sperm ducts formed sperm bundles and initially swam backward with asymmetric flagellar waveforms to detach from the bundles. One hour later, the sperm began to swim forward and in a circle. After an additional 1 h incubation, the sperm swam straight, with a change in the flagellar waveforms from asymmetric to symmetric. Spontaneous backward swimming with symmetric waveforms was also observed. The eusperm stored in the female seminal receptacle were motile and showed forward symmetric swimming with spontaneous backward swimming, which appeared necessary for detachment from the wall of receptacle. All of these motility changes were observed in the absence of parasperm, suggesting that these changes autonomously occur in eusperm. Our waveform analysis of these swimming patterns revealed that only the swimming with symmetric waveform showed reverse propagation of the flagellar waveforms. Both types of backward swimming were diminished in Ca(2+)-free seawater and in seawater containing Ni(2+), indicating the regulation of swimming direction by Ca(2+)-dependent signal transduction.

Testicular testosterone: Estradiol ratio in domestic cats and its relationship to spermatogenesis and epididymal sperm morphology

The phenomenon of teratozoospermia in felids is not fully understood. In this study, we investigated the testicular androgen:estrogen balance in domestic cats and correlated these data with epididymal sperm morphology and the degree of spermatogenic activity. During spring and summer, testes and blood samples were obtained from 37 mixed-breed domestic cats (12 to 48 mo). The epididymal sperm were harvested and evaluated for sperm counts, motility, and morphology. Distal cytoplasmic droplets were not considered a defect, and samples were considered normozoospermic if they contained more than 60% normal sperm (N = 25) or teratozoospermic if they contained less than 45% normal sperm (N = 12). The testicular and serum concentrations of testosterone (T) and 17β-estradiol (E2) were determined with an enzyme immunoassay. The gonadosomatic index and epididymal sperm numbers and motility did not differ between groups. The percentage of normal sperm was higher in normozoospermic (74.3 ± 2.0, mean ± SEM) than in teratozoospermic samples (43.1 ± 1.4). The most prevalent sperm defects in the teratozoospermic group were abnormal acrosomes (9.7 ± 2.0) and bent midpieces (12.2 ± 2.0) or tails (24.0 ± 2.7) with cytoplasmic droplets. Histomorphometric data were similar between groups, although there was a lower Leydig cell nuclear volume in teratozoospermic samples. Normozoospermic samples contained a higher percentage of haploid cells and had a higher index of total spermatogenic transformation than teratozoospermic samples. Serum concentrations of T (0.5 ± 0.1 vs. 0.8 ± 0.4 ng/mL) and E2 (9.5 ± 1.2 vs. 11.4 ± 2.3 pg/mL) and testicular T concentrations (471.6 ± 65.3 vs. 313.4 ± 57.6 ng/g) were similar between groups. However, compared with normozoospermic samples, teratozoospermic samples had higher testicular E2 concentrations (8.5 ± 3.6 vs. 5.4 ± 0.5 ng/g) and a lower T:E2 ratio (31.8 ± 4.1 vs. 87.2 ± 11.6). There were significant correlations between testicular E2 values and percentages of normal sperm (r = −0.55) as well as those with primary sperm defects (r = 0.58) or abnormal acrosomes (r = 0.64). The T:E2 ratio was also correlated with meiotic index (r = 0.45) and percentage of normal sperm (r = 0.58). In conclusion, a high testicular E2 concentration and a reduced T:E2 ratio were significantly associated with higher ratios of abnormal sperm types, suggesting that the balance between androgens and estrogens is an important endocrine component in the genesis of teratozoospermia in felids.

Age‐dependent trade‐offs between immunity and male, but not female, reproduction

Immune function is costly and must be traded off against other life-history traits, such as gamete production. Studies of immune trade-offs typically focus on adult individuals, yet the juvenile stage can be a highly protracted period when reproductive resources are acquired and immune challenges are ubiquitous. Trade-offs during development are likely to be important, yet no studies have considered changes in adult responses to immune challenges imposed at different stages of juvenile development. By manipulating the timing of a bacterial immune challenge to the larvae of the cotton bollworm moth, we examined potential trade-offs between investment into immunity at different stages of juvenile development (early or late) and subsequent adult reproductive investment into sperm or egg production. Our data reveal an age-dependent trade-off between juvenile immune function and adult male reproductive investment. Activation of the immune response during late development resulted in a reduced allocation of resources to eupyrene (fertilizing) sperm production. Immune activation from the injection procedure itself (irrespective of whether individuals were injected with an immune elicitor or a control solution) also caused reproductive trade-offs; males injected early in development produced fewer apyrene (nonfertilizing) sperm. Contrary to many other studies, our study demonstrates these immune trade-offs under ad libitum nutritional conditions. No trade-offs were observed between female immune activation and adult reproductive investment. We suggest the differences in trade-offs observed between male sperm types and the absence of reproductive trade-offs in females may be the result of ontogenetic differences in gamete production in this species. Our data reveal developmental windows when trade-offs between immune function and gametic investment are made, and highlight the importance of considering multiple developmental periods when making inferences regarding the fundamental trade-offs expected between immune function and reproduction.

Directional Freezing of Reproductive Cells and Organs

Directional freezing is based on a simple thermodynamic principle where ice crystals are precisely controlled through the sample by regulating the velocity of the sample movement through the predetermined temperature gradient. Directional freezing permits a precise and uniform cooling rate in both small and large volume samples. Directional freezing was used for slow and rapid freezing, as well as for vitrification of oocytes and embryos using the minimum drop size technique. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The method enabled, for the first time, successful freezing of a whole ovary and freeze-drying of mammalian cells followed by thawing and transplantation and rehydration, respectively.

Identification of differentially expressed proteins between bull X and Y spermatozoa

Differential expression of genes leads to variation in phenotypes of X and Y sperm, even though some differential gene products are shared through an intercellular bridge. Differentially expressed proteins between X and Y sperm sorted from semen of nine bulls were compared using two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis. Overall, 663±12 and 647±22 protein spots were detected in X sperm and Y sperm, respectively, and 42 significant protein spots were differentially expressed between them (P<0.05). Sixteen of these protein spots were successfully identified by MS and tandem MS and were found to be closely relevant to energy metabolism, stress resistance, cytoskeletal structure and the activity of serine proteases. Expression levels of two of these proteins, CAPZB and UQCRC1, were verified by Western blot. We propose that these differentially expressed proteins may affect the phenotype of X and Y sperm, binding and fusion of sperm/oocyte and development of the zygotic embryo. Our preliminary results provide an overview of differential expression in total protein levels between X and Y spermatozoa. Identification of these altered proteins may provide a theoretical basis for understanding the biological differences between the two types of sperm.

Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Optimizing the use of sex-sorted sperm in timed artificial insemination programs for suckled beef cows1

Three experiments were designed to evaluate methods to optimize the use of sex-sorted sperm in timed AI (TAI) programs for suckled beef cows. In all 3 experiments, suckled Bos indicus cows were synchronized using an intravaginal progesterone (P4) device during 8 d and a 2.0-mg injection of intramuscular estradiol benzoate (EB) at device insertion. The females received PG and eCG (300 IU) at P4 device removal and 1.0 mg of EB 24 h later. The cows were inseminated 60 to 64 h after P4 device withdrawal. All cows had their ovaries scanned by transrectal ultrasound at TAI to indentify and to measure the largest follicle (LF) present. In Exp. 1, a total of 853 cows had their LF classified as <9 mm or ≥9 mm at the time of TAI; these cows were then randomly assigned to 4 groups according to their LF diameter (<9 mm or ≥9 mm) and the type of sperm used (sex-sorted or non-sex-sorted). There was an interaction (P = 0.02) between the type of sperm and LF diameter beginning at TAI[non-sex-sorted ≥9 mm = 58.9%a (126/214); non-sex-sorted <9 mm = 49.5%b (106/214);sex-sorted ≥9 mm = 56.8%ab (134/236); and sex-sorted <9 mm = 31.2%c (59/189), a≠b≠c = P < 0.05]. In Exp. 2, suckled cows (n = 491) were classified immediately before TAI as having displayed estrus or not (estrus or no estrus) between P4 device removal and TAI. These cows were randomly assigned to 4 groups according to the occurrence of estrus and the type of sperm (sex-sorted or non-sex-sorted). There were effects of the occurrence of estrus (P = 0.0003) and the type of sperm (P = 0.05) on pregnancy per AI [P/AI; no estrus, non-sex-sorted = 43.6% (27/62); estrus, non-sex-sorted = 58.5%; (107/183); no estrus, sex-sorted = 33.9% (21/62), and estrus, sex-sorted = 50.0% (92/184)]; however, no interaction between the occurrence of estrus and type of sperm was observed (P = 0.87). In Exp. 3, a total of 200 suckled cows presenting LF ≥9 mm at TAI were randomly assigned to receive sex-sorted sperm deposited into the uterine body (n = 100) or into the uterine horn ipsilateral to the recorded LF (n = 100). No effect of deeper AI on P/AI was found (P = 0.57). Therefore, the LF diameter at TAI and the occurrence of estrus can be used as selection criteria to identify cows with greater odds of pregnancy to receive sex-sorted sperm in TAI programs. In addition, performing TAI with sex-sorted sperm deeper into the uterus did not alter the pregnancy results.

Aquaporins in sperm osmoadaptation: an emerging role for volume regulation

Upon ejaculation, mammalian sperm experience a natural osmotic decrease during male to female reproductive tract transition. This hypo-osmotic exposure not only activates sperm motility, but also poses potential harm to sperm structure and function by inducing unwanted cell swelling. In this physiological context, regulatory volume decrease (RVD) is the major mechanism that protects cells from detrimental swelling, and is essential to sperm survival and normal function. Aquaporins are selective water channels that enable rapid water transport across cell membranes. Aquaporins have been implicated in sperm osmoregulation. Recent discoveries show that Aquaporin-3 (AQP3), a water channel protein, is localized in sperm tail membranes and that AQP3 mutant sperm show defects in volume regulation and excessive cell swelling upon physiological hypotonic stress in the female reproductive tract, thereby highlighting the importance of AQP3 in the postcopulatory sperm RVD process. In this paper, we discuss current knowledge, remaining questions and hypotheses about the function and mechanismic basis of aquaporins for volume regulation in sperm and other cell types.

Cryptic gametic interactions confer both conspecific and heterospecific advantages in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone

Most species pairs are isolated through the collective action of a suite of barriers. Recent work has shown that cryptic barriers such as conspecific sperm precedence can be quite strong, suggesting that they evolve quickly. However, because the strength of multiple barriers has been formally quantified in very few systems, the relative speed with which conspecific sperm precedence evolves remains unclear. Here, we measure the strength of both conspecific sperm precedence and cryptic non-competitive isolation between the hybridizing sister species, Chrysochus auratus and C. cobaltinus (Coleoptera: Chrysomelidae), and compare the strength of those barriers to the strength of other known reproductive barriers in this system. Overall, cryptic barriers in this system are weaker than other barriers, indicating that they have not evolved rapidly. Furthermore, their evolution has been asymmetric. Non-competitive barriers substantially reduce the production of hybrid offspring by C. auratus females but not by C. cobaltinus females. In multiply-mated C. cobaltinus females, heterospecific sperm outcompete conspecific sperm, as evidenced by the fact that heterospecific males sired disproportionately more offspring than predicted from the results for singly-mated females. In C. auratus females, neither sperm type has a competitive advantage. Such asymmetries explain why nearly all F1 hybrids in the field are from crosses between C. cobaltinus females and C. auratus males. We discuss these findings in terms of understanding the cost of mating 'mistakes' in the Chrysochus hybrid zone. In addition, our discovery that 95% confidence intervals for commonly-used isolation statistics can be very wide has important implications for speciation research. Specifically, to avoid biases in the interpretation of such isolation metrics, we suggest that studies should routinely include error estimates in their analyses of reproductive isolation.

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml −1) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca 2+-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased ( P < 0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh ( P < 0.01) and frozen-thawed ( P < 0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca 2+-influxes were also significantly stimulated by relaxin in the fresh ( P < 0.01) and frozen-thawed ( P < 0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh ( P < 0.01) and frozen-thawed ( P < 0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca 2+-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.