Sperm mobility determines the outcome of sperm competition in the domestic fowl

The aim of this study was to establish whether the mobility of sperm of the domestic fowl, as measured by an in vitro assay, predicted the outcome of sperm competition. Thirteen pairs of New Hampshire roosters, comprising one male categorized as having high-mobility sperm and the other as having average-mobility sperm, were used. Each male provided 25 x 10(6) sperm, which were mixed and artificially inseminated into between four and seven New Hampshire hens, each of which produced 2-11 offspring. The experiment was conducted twice, such that the same pair of males inseminated the same females. Paternity was assigned by using microsatellite markers. There was a clear effect of sperm-mobility phenotype on the outcome of sperm competition: in all 13 pairs the high-mobility male fathered the majority of offspring (75.3% overall; p < 0.0001). The proportion of offspring fathered by the high-mobility male within pairs varied significantly between male pairs (p < 0.0005). This effect was associated with the difference in sperm-mobility scores between males within pairs; there was a significant positive relationship between the proportion of offspring fathered by the high-mobility male and the ratio of mobility scores between males (p < 0.05). In addition, compared with their success predicted from the non-competitive situation, in the competitive situation high-mobility males were disproportionately successful in fertilizing eggs compared with average-mobility males. This may occur because female sperm storage is limited in some way and a greater proportion of high-mobility sperm gain access to the female's sperm storage tubules. There was no evidence that female effects accounted for any of the variation in paternity.     

Sperm mobility: phenotype in roosters (Gallus domesticus) determined by mitochondrial function

Previously, inheritance of sperm mobility entailed a maternal additive genetic effect, and sperm ATP content was correlated (r = 0.80) with phenotype. The present study was conducted to determine if mitochondrial function was critical to phenotypic expression. Whereas phenotype was independent of mitochondrial helix length, phenotype was correlated with sperm oxygen consumption (r = 0.83) using random-bred roosters. Aberrant mitochondria characterized immobile sperm, as evidenced by transmission-electron microscopy. Such mitochondria were swollen and contained disorganized cristae. Additional experiments were performed with roosters from lines selected for low or high sperm mobility. A threefold difference in sperm oxygen consumption was observed between lines. Single nucleotide polymorphisms were observed in mitochondrial DNA by sequencing replicate mitochondrial genomes from each line. An A-to-G substitution in the gene encoding tRNA(Arg) was inherited consistently, as evidenced by restriction fragment length polymorphism analysis using two male and two female progeny per family group and 14 family groups per line. Motile concentration in semen from low-line males was half that observed in semen from high-line males, as evidenced by computer-assisted sperm motion analysis. Likewise, 47% of sperm from low-line males contained aberrant mitochondria, compared to 4% for high-line males. In summary, sperm mobility phenotype was dependent on mitochondrial function, which in turn was altered by genetic selection. Fowl deferent duct fluid contains a high concentration of glutamate. We propose that variation in sperm mobility phenotype stems from the extent to which glutamate induces excessive mitochondrial Ca2+ uptake before ejaculation.     

Sperm mobility: phenotype in roosters (Gallus domesticus) determinedby concentration of motile sperm and straight line velocity

Previous research demonstrated that sperm mobility, i.e., the net movement of a sperm population, is a quantitative trait of the domestic fowl. However, the cellular basis for this trait was unknown. In the present work, individual motile sperm were evaluated with a Hobson SpermTracker in order to identify one or more properties of motile sperm that could account for variation in sperm mobility observed among males. A method was validated for assessing sperm motion over an erythrocyte monolayer at body temperature. A small-scale experiment with roosters from the tails and center of a normal distribution of sperm mobility phenotypes (n = 33 roosters) demonstrated that straight line velocity (VSL) and motile concentration were critical to expression of phenotype. The importance of these variables was confirmed with a large-scale experiment using a representative subpopulation (n = 100 roosters). VSL of individual sperm at 41 degrees C ranged between 5 and 100 microm/sec. VSL averaged 32, 39, and 40 microm/sec for low, average, and high sperm mobility phenotypes. Sperm were diluted to 1.2 x 10(6)/ml for motion analysis. Mean motile concentrations were 0.52, 0.84, and 0.95 x 10(6)/ml for low, average, and high sperm mobility phenotypes. Motile concentration was correlated with sperm mobility (r = 0.71). VSL appeared to have an additive effect as it was correlated with straightness of sperm cell trajectory (r = 0.79).     

Between‐male variation in sperm size,velocity and longevity in sand martins Riparia riparia

Sperm mobility is known to be an important determinant of a male's sperm competitive ability. Although more debated, sperm length and its relation to sperm swimming ability has also been proposed to determine a male's fertilisation potential. Furthermore, both mobility and length may covary with a male's phenotype, either positively (the phenotype‐linked fertility hypothesis) or negatively if, for instance, low‐quality males have less access to females but invest more in sperm production. Using dummy females, we collected sperm samples from wild sand martins Riparia raparia males. We investigated the relationship between sperm length and sperm swimming speed as measured by sperm straight line velocity (VSL), and determined whether sperm traits are correlated with male body size and condition. We found that total sperm length is repeatable within‐ejaculate and shows substantial inter‐male variation. Sperm length was associated with sperm velocity: males with short sperm have sperm that swim initially faster but die sooner, whereas males with longer sperm have sperm that swim more slowly but for a longer time. Smaller males produced sperm with higher overall velocity. This correlation between male size and sperm behaviour may reflect alternative fertilisation strategies where small males having less mating opportunities invest more in sperm competitive ability. The existence of such alternative strategies would participate in maintaining variation in sperm length and velocity in this species.     

Turkey sperm mobility influences paternity in the context of competitive fertilization.

We have devised a novel means of investigating competitive fertilization in turkeys, using microsatellite genotyping to identify male parentage. Our results demonstrate that sperm mobility is a mechanism responsible in part for paternity efficiency in turkeys. Sperm mobility is composed of several parameters in which sperm motility is a component. Differences between ejaculates in the number of sperm penetrating into a dense, insert, nontoxic solution were measured and used to classify males into high, average, or low sperm mobility phenotypes. Microsatellite genotyping was used to determine parentage of poults after equal numbers of sperm from 10 males (either high or average phenotype, n = 5, mixed with low phenotype, n = 5) were inseminated simultaneously. In a separate study, the numbers of sperm hydrolyzing the perivitelline layer of eggs were compared between hens inseminated with sperm from high-, average-, or low-phenotype males. Overall, heterospermic inseminations resulted in consistently fewer offspring produced by low-mobility phenotype males. This correlated with physiological data in which semen from the low-mobility males had reduced numbers of sperm at the fertilization site as determined by sperm hole counts in the perivitelline layer of eggs. This is the first illustration of a measurable sperm trait predictive of paternity success in a competitive fertilization trial in turkeys, a species that is predominately reproduced by artificial insemination of multiple-sire pools.     

Sperm head phenotype and male fertility in ram semen

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter²/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.     

Sperm mobility: deduction of a model explaining phenotypic variation in roosters (Gallus domesticus)

In previous work, variation in sperm mobility phenotype was attributed to the proportion of ejaculated fowl sperm containing dysfunctional mitochondria. In the present work, latent mitochondrial dysfunction was inferred from patterns of sperm egress from the oviduct's sperm-storage tubules. In addition, experiments were performed to help explain how mitochondrial function could be compromised in viable sperm cells. Confocal microscopy demonstrated that sperm Ca2+ content differed between low and high sperm-mobility phenotypes when sperm were stained with rhod-2 AM, a Ca2+ -specific dye. Fluorescence was associated with the nuclear envelope, a variant of the endoplasmic reticulum, and greater fluorescence was observed in sperm from low sperm-mobility males. Fluorescence was reduced by 50% when motile sperm were rendered immotile by incubation with a Ca2+ chelator. Thus, a relationship was established between a dynamic intracellular Ca2+ pool and sperm motility. Sperm N-methy-D-aspartic acid (NMDA) receptors were inferred by the action of D-homocysteinesulfinic acid, a potent NMDA receptor agonist. Seminal plasma from low sperm mobility males was characterized by an elevated glutamate concentration. Thapsigargin, which inhibits the smooth endoplasmic reticulum Ca2+ pump and thereby promotes Ca2+ efflux, rendered sperm immotile. This effect was blocked by cyclosporin A, which prevents the formation of the mitochondrial permeability transition pore (PTP) in response to elevated mitochondrial Ca2+ content. In summary, we propose that 1) glutamate enables Ca2+ uptake into sperm before ejaculation, 2) excessive Ca2+ uptake triggers formation of the PTP in a subpopulation of sperm, and 3) sperm mobility is decreased in proportion.     

Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract

The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm². Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract.     

Induction of immunity to spermatozoa in male domestic fowl and effects on fertility.

Male fowl were immunized intravenously (i.v.) or intramuscularly (i.m.) with spermatozoa to assess the effects of immunity to spermatozoa on fertility. Histological and immunofluorescence evaluations of testis and ductus deferens tissues after 24 weeks of immunizations revealed immune cell infiltration and immunoglobulin associated with spermatozoa. The long-term immunization regimen resulted in significant antisperm antibody titres in the immunized groups. When semen from i.v.-immunized males was used to inseminate females, fertility over 7 days was reduced (P less than 0.05). A subsequent experiment using a 10-week i.v. immunization scheme also led to high antisperm titres. Spermatozoa from these males were characterized by lower fertility and duration of fertility than those of controls (P less than 0.05). As in mammals, a reduction in fertility may result from exposure of avian males to sperm antigens.     

Do male secondary sexual characters signal ejaculate quality? A meta‐analysis

There are two reasons why researchers are interested in the phenotypic relationship between the expression of male secondary sexual characters (SSCs) and ‘ejaculate quality’ (defined as sperm/ejaculate traits that are widely assumed to increase female fertility and/or sperm competitiveness). First, if the relationship is positive then females could gain a direct benefit by choosing more attractive males for fertility assurance reasons (‘the phenotype‐linked fertility’ hypothesis). Second, there is much interest in the direction of the correlation between traits favoured by pre‐copulatory sexual selection (i.e. affecting mating success) and those favoured by post‐copulatory sexual selection (i.e. increasing sperm competitiveness). If the relationship is negative this could lead to the two forms of selection counteracting each other. Theory predicts that the direction of the relationship could be either positive or negative depending on the underlying genetic variance and covariance in each trait, the extent of variation among males in condition (resources available to allocate to reproductive traits), and variation among males in the cost or rate of mating. We conducted a meta‐analysis to determine the average relationship between the expression of behavioural and morphological male secondary sexual characters and four assays of ejaculate quality (sperm number, viability, swimming speed and size). Regardless of how the data were partitioned the mean relationship was consistently positive, but always statistically non‐significant. The only exception was that secondary sexual character expression was weakly but significantly positively correlated with sperm viability (r = 0.07, P < 0.05). There was no significant difference in the strength or direction of the relationship between behavioural and morphological SSCs, nor among relationships using the four ejaculate quality assays. The implications of our findings are discussed.     

Ejaculate investment and attractiveness in the stalk‐eyed fly,Diasemopsis meigenii

The phenotype‐linked fertility hypothesis proposes that male fertility is advertised via phenotypic signals, explaining female preference for highly sexually ornamented males. An alternative view is that highly attractive males constrain their ejaculate allocation per mating so as to participate in a greater number of matings. Males are also expected to bias their ejaculate allocation to the most fecund females. We test these hypotheses in the African stalk‐eyed fly, Diasemopsis meigenii. We ask how male ejaculate allocation strategy is influenced by male eyespan and female size. Despite large eyespan males having larger internal reproductive organs, we found no association between male eyespan and spermatophore size or sperm number, lending no support to the phenotype‐linked fertility hypothesis. However, males mated for longer and transferred more sperm to large females. As female size was positively correlated with fecundity, this suggests that males gain a selective advantage by investing more in large females. Given these findings, we consider how female mate preference for large male eyespan can be adaptive despite the lack of obvious direct benefits.     

Male Mating History Influences Female Mate Choice in the Trinidadian Guppy (Poecilia reticulata)

Based on the phenotype‐linked fertility hypothesis, sexual selection should favour females that can accurately assess the recent mating history of available sexual partners and preferentially avoid mating with recently‐mated males [who may be sperm depleted (SD)] so as to minimize the risk of their eggs not being fertilized. This hypothesis has received to date only limited attention and empirical support. Therefore, in the current study, we investigated experimentally whether females of a vertebrate species, the Trinidadian guppy (Poecilia reticulata), are able to assess the recent mating history of males, and thus potentially their functional fertility, and choose to avoid mating with males that appear to have recently mated and who may be sperm limited. Individual virgin females were first given a dichotomous choice between a male that had not been recently observed to interact sexually with another female (i.e. not sperm‐depleted) and another male that had been observed to interact sexually with a female (i.e. potentially sperm‐depleted) as sexual partners. Paired males were matched for body length and coloration. Immediately following this test, the focal females were subjected to a free‐swimming mate‐choice test using the same paired stimulus males. As predicted, on average, female guppies avoided the apparently recently‐mated (and potentially sperm‐depleted) male and exhibited a significant preference for the other male not recently observed mating (and thus not likely sperm limited) during both tests. We do not yet fully understand the underlying mechanisms of this preference. Therefore, further research on the particular cues that females use to assess the recent mating history and fertility status of males is required.     

Melanism in a Chinese Population of Harmonia axyridis (Coleoptera: Coccinellidae): A Criterion for Male Investment with Pleiotropic Effects on Behavior and Fertility

In Beijing, China, females of Harmonia axyridis are promiscuous but prefer typical (succinea form) males to melanic ones in the spring generation, ostensibly due to the thermal disadvantages of melanism during summer. We used laboratory observations to test whether males invested differentially in females according to their elytral color, and whether male behavior was phenotype-dependent. Video-recording was used to monitor no-choice mating tests between virgin adults in all phenotype combinations and females were isolated post-copula to observe their egg retention times and reproduction over 5 days. Females tended to wait longer before using the sperm of melanic males, and melanic females delayed longer than succinic females. Melanic males spent longer in copula with succinic than melanic females and the latter received fewer bouts of male abdominal shaking that correlate with sperm transfer, regardless of the phenotype of their mate. Although melanic males abandoned melanic females faster than did succinic males, they remained in copula with females of both phenotypes for a longer period after shaking, suggesting a larger investment in mate guarding by the less-preferred male phenotype. Although female fecundity did not vary among phenotype combinations, egg fertility was lower for females mated to melanic males, suggesting a pleiotropic effect of melanism on male fertility in addition to its effects on male mating behavior.     

Methods for Cryopreservation of Guinea Fowl Sperm

Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet) and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide). The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining). Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols (based on in vitro assessment of post-thaw semen quality) were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively). During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.     

SNARE proteins and caveolin-1 in stallion spermatozoa: possible implications for fertility

Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobrevin/VAMP, the calcium sensor synaptotagmin, and the ATPase NSF. Also present is caveolin-1, a component of lipid rafts. Stallions with fertility problems presented the worst quality of sperm and acrosomal membrane, and had less sperm cells stained positively for SNAREs and caveolin-1, than sperm from fertile donors (p < 0.001). Ubiquitin surface staining was also performed and it seemed to inversely correlate with stallion fertility, supporting data obtained with the negative staining technique. A male-related problem was confirmed when mares that had failed to impregnate with samples from an infertile stallion were successfully inseminated with sperm from a fertile donor. Furthermore NSF, synaptotagmin and caveolin-1 staining seemed to be useful in predicting stallion fertility, i.e. significantly more sperm cells stained positively for these proteins in samples from fertile males. Although these results need to be expanded on a larger scale, they suggest that acrosomal and surface staining of equine sperm with novel probes may constitute useful tools in predicting stallion fertility.     

Intracytoplasmic Sperm Injection Using DNA-Fragmented Sperm in Mice Negatively Affects Embryo-Derived Embryonic Stem Cells,Reduces the Fertility of Male Offspring and Induces Heritable Changes in Epialleles

Intracytoplasmic sperm injection (ICSI) in mice using DNA-fragmented sperm (DFS) has been linked to an increased risk of genetic and epigenetic abnormalities both in embryos and offspring. This study examines: whether embryonic stem cells (ESCs) derived from DFS-ICSI embryos reflect the abnormalities observed in the DFS-ICSI progeny; the effect of DFS-ICSI on male fertility; and whether DFS-ICSI induces epigenetic changes that lead to a modified heritable phenotype. DFS-ICSI-produced embryos showed a low potential to generate ESC lines. However, these lines had normal karyotype accompanied by early gene expression alterations, though a normal expression pattern was observed after several passages. The fertility of males in the DFS-ICSI and control groups was compared by mating test. Sperm quantity, vaginal plug and pregnancy rates were significantly lower for the DFS-ICSI-produced males compared to in vivo-produced mice, while the number of females showing resorptions was higher. The epigenetic effects of DFS-ICSI were assessed by analyzing the phenotype rendered by the Axin1^Fu allele, a locus that is highly sensitive to epigenetic perturbations. Oocytes were injected with spermatozoa from Axin1^{Fu /+} mice and the DFS-ICSI-generated embryos were transferred to females. A significantly higher proportion of pups expressed the active kinky-tail epiallele in the DFS-ICSI group than the controls. In conclusion: 1) ESCs cannot be used as a model of DFS-ICSI; 2) DFS-ICSI reduces sperm production and fertility in the male progeny; and 3) DFS-ICSI affects the postnatal expression of a defined epigenetically sensitive allele and this modification may be inherited across generations.     

Sperm mobility: mechanisms of fertilizing efficiency, genetic variation and phenotypic relationship with male status in the domestic fowl, Gallus gallus domesticus

When females are sexually promiscuous, sexual selection continues after insemination through sperm competition and cryptic female choice, and male traits conveying an advantage in competitive fertilization are selected for. Although individual male and ejaculate traits are known to influence paternity in a competitive scenario, multiple mechanisms co-occur and interact to determine paternity. The way in which different traits interact with each other and the mechanisms through which their heritability is maintained despite selection remain unresolved. In the promiscuous fowl, paternity is determined by the number of sperm inseminated into a female, which is mediated by male social dominance, and by the quality of the sperm inseminated, measured as sperm mobility. Here we show that: (i) the number of sperm inseminated determines how many sperm reach the female sperm-storage sites, and that sperm mobility mediates the fertilizing efficiency of inseminated sperm, mainly by determining the rate at which sperm are released from the female storage sites, (ii) like social status, sperm mobility is heritable, and (iii) subdominant males are significantly more likely to have higher sperm mobility than dominant males. This study indicates that although the functions of social status and sperm mobility are highly interdependent, the lack of phenotypic integration of these traits may maintain the variability of male fitness and heritability of fertilizing efficiency.     

High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing

Hybrid catfish created by crossing of female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: (1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; (2) evaluate the effect of sperm concentration on cryopreservation; (3) refine cryoprotectant concentration based on the highest effective sperm concentration; (4) compare the effect of thawing samples at 20 or 40 °C; (5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; (6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and (7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5 °C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1 × 10⁹ sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20 °C for 40 s or 40 °C for 20 s. After fertilization, the percentage of neurulation (Stage V embryos) was 80 ± 21%, and percentage of embryonic mobility (Stage VI embryo) was 51 ± 22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm (P = 0.010) and channel catfish sperm (P = 0.023), but not for Stage VI embryos (P ? 0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species.     

Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls

We investigated fluorometry to study sperm viability and flow cytometry to study sperm chromatin structure. We also assessed sperm quality after thawing relative to field fertility after AI as shown by 56-day non-return rates (56-d NRR) Frozen-thawed semen samples were obtained from 20 Swedish Red and White bulls (1 to 3 semen batches/bull) and the fertility data were based on 6,369 AIs. Fluorometry enabled simultaneous detection of sperm viability and concentration in Hoechst 33258-stained semen samples. Sperm chromatin structure assay (SCSA) evaluated denaturability of sperm nuclear DNA in situ after acid treatment. The intensity of fluorescence in non-permeabilized samples was negatively (r = -0.60, P < 0.001) correlated with microscopically-assessed sperm viability, and the fluorescence of permeabilized semen samples significantly (r = 0.67, P < 0.001) correlated with sperm concentration as assessed by hemocytometry. From the fluorescence output, the calculated percentage of damaged cells was negatively (r = -0.71, P < 0.001) correlated with the number of live cells derived from the microscopic assessment of sperm viability and concentration. This variable was significantly correlated with fertility results both at batch (r = -0.39, P < 0.05), and bull (r = -0.57, P < 0.01) levels. The SCSA variables SDalphat and COMPalphat were significantly (r = -0.59-0.64, P < 0.001) correlated with sperm viability variables after thawing but only the COMPalphat correlated significantly (r = -0.53, P < 0.05) with fertility results and solely at the bull level. The results indicate that fluorometric assessment is in good agreement with other practiced procedures and can be performed with sufficient accuracy. The SCSA may be a valuable complement for routinely practiced microscopic evaluation of sperm morphology of AI bull semen     

The structural organization of sperm chromatin

The structure of rabbit, fowl, and Xenopus laevis sperm chromatin was explored by study of the reaction of their decondensed nuclei with DNase 1 and micrococcal nuclease. Those of rabbit and fowl were readily digested by DNase 1, and the polyacrylamide gel electrophoresis profiles of DNAs extracted from the digests were similar, each being polydisperse with a single discrete band of DNA smaller than 72 base pairs. There were differences, however, between the sperm chromatins in the course of their digestion by micrococcal nuclease. A limit digest at about 45% acid solubility was obtained with Xenopus sperm chromatin, while 90% of fowl sperm DNA was rendered acidsoluble by the enzyme. The gel profiles of the limit digests were polydisperse, but only those of rabbit and fowl sperm chromatins possessed a discrete band of DNA smaller than 72 base pairs. Bleomycin did not react with DNA of rabbit, fowl, or Xenopus spermatozoa. Since bleomycin reacts with somatic cell chromatin, and the course of DNase 1 or micrococcal nuclease digestion of sperm chromatin was different from that found for somatic cell chromatin, it would appear that sperm chromatin does not have the repeating nucleosometype structure of somatic cell chromatin. The nuclease digestion studies further suggest that the organization of rabbit and fowl sperm chromatins is similar, and is different from that of Xenopus sperm chromatin. The dependence of the structure of sperm chromatin on the composition of its basic proteins, and a possible structure for a protamine-type sperm chromatin, are discussed.