The duration of ovulation in pigs, studied by transrectal ultrasonography, is not related to early embryonic diversity

The duration of ovulation in pigs was studied by transrectal ultrasonography. The number of preovulatory follicles was counted on both ovaries at 30-minute intervals from 36 hours after the onset of estrus (Group A: naturally ovulating sows that were group-housed and were inseminated and caged during scanning) or 40 hours after treatment with human chorionic gonadotropin (hCG) (Group B: tethered sows that had been induced to ovulate but were not inseminated). The duration of ovulation was (mean+/-SD) 1.8+/-0.6 hours (range 0.75 to 3.25) in Group A (n=13) and 4.6+/-1.7 hours (range 2.0 to 7.0) in Group B (n=8). The difference was significant (P<0.01). In Group A and B sows, respectively, the course of ovulation, expressed as the relation between the relative follicle count (percentage of the maximum follicle count; Y) and the time (percentage of the duration of ovulation; X) was: Y = 104.3( *)e(-0.023( *)X) (R(2)=0.95) and Y = 98.9( *)e(-0.018( *)X) (R(2)=0.92). The onset of ovulation occurred at approximately two-thirds of the duration of the estrus (Group A: 67+/-6%; Group B: 60+/-10%). Group A sows were artificially inseminated and were slaughtered at 98+/-8 hours (range 77 to 110) after ovulation. The difference between the maximum follicle count and the corpora lutea count was zero or only 1 in 81% (21 26 ) of the ovaries. Embryonic diversity (within-litter SD of the number of nuclei or of the number of cell cycles) was not related to the duration of ovulation, neither at the level of ovary nor of sow (P>0.05). In conclusion, transrectal ultrasonography was found to be an appropriate nonsurgical method of studying the duration of ovulation in pigs. The duration of ovulation varied both between sows and between groups of sows, and was not related to early embryonic diversity.     

In synchronized pigs, the duration of ovulation is not affected by insemination and is not a determinant for early embryonic diversity

The duration of ovulation (i.e., the time interval between the ovulation of the first and the last follicle in a sow during an estrus) is said to be related to embryonic diversity, which in turn is related to embryonic mortality. The relationship between the duration of ovulation and within-litter early embryonic diversity and the influence of insemination on the duration of ovulation were studied. To determine whether ovulation assessment (transrectal ultrasonography) influenced early embryonic development, control sows were not scanned. Multiparous cyclic sows with an exogenously induced estrus were used. Ovulation detection by means of transrectal ultrasonography did not influence fertilization rate, accessory sperm count, early embryonic development or early embryonic diversity, and, therefore, ultrasonography appears to be a worthwhile method for studying the time and duration of ovulation. Insemination did not influence (P>0.05) the duration of ovulation, in sows which ovulated between 39 and 49 hours after hCG injection. The duration of ovulation (mean+/-SD(range)) was 2.4+/-0.7 (1.1 to 4.0) hours in 15 sows which were artificially inseminated at 22 and 30 hours after hCG injection. In 8 noninseminated sows, the duration of ovulation was 2.3+/-0.5 (1.5 to 3.3) hours. The duration of ovulation was not related to embryonic diversity (SD of the number of nuclei or the number of cell cycles of embryos in a litter) at 114 to 121 hours after ovulation. Thus, a difference of up to 3 hours in the duration of ovulation does not seem to be an important determinant of variation in embryonic diversity between sows.     

Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds

Spermatozoa are among the most diversified cells in the animal kingdom, but the underlying evolutionary forces affecting intraspecific variation in sperm morphology are poorly understood. It has been hypothesized that sperm competition is a potent selection pressure on sperm variation within species. Here, we examine intraspecific variation in total sperm length of 22 wild passerine bird species (21 genera, 11 families) in relation to the risk of sperm competition, as expressed by the frequency of extrapair paternity and relative testis size. We demonstrate, by using phylogenetic comparative methods, that between-male variation in sperm length within species is closely and negatively linked to the risk of sperm competition. This relationship was even stronger when only considering species in which data on sperm length and extrapair paternity originated from the same populations. Intramale variation in sperm length within species was also negatively, although nonsignificantly, related to sperm competition risk. Our findings suggest that postcopulatory sexual selection is a powerful evolutionary force reducing the intraspecific phenotypic variation in sperm-size traits, potentially driving the diversification of sperm morphology across populations and species.     

The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content

The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Since reduction of sperm nuclear disulfide (S-S) bonds is a prerequisite for sperm nuclear decondensation in vitro and in vivo, we hypothesized that sperm nuclei with relatively few S-S bonds would require less time to decondense in the oocyte than sperm nuclei with higher numbers of S-S bonds, and that male pronucleus formation would occur more rapidly as well. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, were microinjected into hamster oocytes, and the time course of sperm nuclear decondensation and male pronucleus formation was charted. Cauda epididymal sperm nuclei, which are rich in S-S bonds, required 45-60 min to decondense. In contrast, nuclei containing few S-S bonds (namely sonication-resistant spermatid nuclei and cauda epididymal sperm nuclei treated in vitro with the S-S bond-reducing agent dithiothreitol) decondensed within 5-10 min of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 min. Regardless of when decondensation occurred, formation of the male pronucleus never preceded that of the female pronucleus, which occurred 1.25-1.5 h after microinjection. However, sperm nuclei with few S-S bonds were more likely than S-S rich nuclei to transform into male pronuclei in synchrony with the formation of the female pronucleus. We conclude that the timing sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.     

The prenatal maturity of the accessory olfactory bulb in pigs

The morphological development of the accessory olfactory bulb of the fetal pig was studied by classical and histo-chemical methods, and the vomeronasal organ and nasal septum were studied histochemically. Specimens were obtained from an abattoir and their ages estimated from their crown-to-rump length. The accessory olfactory bulb was structurally mature in fetuses of crown-to-rump length 21-23 cm, by which time the lectin Lycopersicum esculentum agglutinin stained the same structures as in adults (in particular, the entire sensory epithelium of the vomeronasal organ, the vomeronasal nerves, and the nervous and glomerular layers of the accessory olfactory bulb). These results suggest that the vomeronasal system of the pig may, like that of vertebrates such as snakes, be functional at birth.     

The signaling protein CD38 is essential for early embryonic development

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. Although a variety of functions have been ascribed to CD38, such as immune responses, insulin secretion, and social behavior in adults, nothing is known of its role during embryonic development when Ca(2+) signals feature prominently. Here, we report the identification and functional expression of CD38 from Xenopus laevis, a key model organism for the study of vertebrate development. We show that CD38 expression and endogenous ADP-ribosyl cyclase activity are developmentally regulated during cellular differentiation. Chemical or molecular inhibition of CD38 abolished ADP-ribosyl cyclase activity and disrupted elongation of the anterior-posterior axis and differentiation of skeletal muscle, culminating in embryonic death. Our data uncover a previously unknown role for CD38 as an essential regulator of embryonic development.     

Antibodies to sperm surface antigens and the c-myc proto-oncogene product inhibit early embryonic development in mice.

The effects of antibodies against sperm antigens and the c-myc proto-oncogene product on early embryonic development were investigated in mice. Affinity-purified Fab' antibodies against lithium diiodosalicylate (LIS)-solubilized murine sperm extract and fertilization antigen (FA-1) reduced (p less than 0.01 to p less than 0.001) blastulation rates of in vitro cultured 2-cell murine embryos primarily because of an arrest of development at the morula stage. Similarly, the c-myc monoclonal antibody (mAb) affected early embryonic development in a dose-dependent manner. These effects were specific, since immunoabsorption, with its respective peptide, completely blocked the inhibitory effect of the c-myc mAb. Anti-LIS sperm Fab' identified four protein bands (approx. 36, 29, 24.6, and 17.6 kDa) on Western blots of extracts from unfertilized and fertilized ova, one band (approx. 68 kDa) each on 4-8-cell embryo and morula extracts, and one band (approx. 53 kDa) on blastocyst extracts. Anti-FA-1 Fab' did not react with unfertilized or fertilized ova, but specifically identified two protein bands (approx. 53 and 25.7 kDa) on blots of 2-cell-embryo extract, one band (approx. 25.7 kDa) on morula extract, and one band (approx. 53 kDa) on blastocyst extract. The c-myc mAb did not react with any band corresponding to the c-myc protein on blots of extracts from unfertilized or fertilized ova, 2-cell embryos, 4-8-cell embryos, morulae, or blastocysts. These results suggest that some of the cross-reacting sperm antigens that are expressed during early cleavages, and the product of the c-myc proto-oncogene may have a role in normal early embryonic development.     

Effects of male accessory sex gland secretions on early embryonic development in the golden hamster

The ventral prostates, dorsolateral prostates, coagulating glands, seminal vesicles and/or ampullary glands were bilaterally removed from adult male hamsters. Removal of these glands did not affect the fertilization rate and cleavage of the embryos at 48 h post coitum (p.c.). Air-dried preparations of the embryos showed a delay in cleavage at 72 h p.c. and a significant number of degenerated embryos was also found in females mated with males from which all the male accessory sex glands had been removed. A significant implantation loss was also observed at 122 h p.c. The results suggest that, in the golden hamster, removal of the male accessory sex gland causes a slower cleavage rate in embryonic development and a significant embryonic loss during pregnancy.     

A whole genome scanning for quantitative trait loci on traits related to sperm quality and ejaculation in pigs

To identify quantitative trait loci (QTL) for traits related to semen and ejaculation, phenotype data including semen volume, sperm concentration, total sperm per ejaculate, sperm motility, sperm abnormality rate, semen pH value, ejaculation times and ejaculation duration were measured on 206 F(2) boar at 240 days in a White Duroc x Erhualian intercross. A genome-wide scan was performed and the entire White Duroc x Erhualian intercross was genotyped for 183 microsatellite markers covering the whole pig genome. QTL analysis was performed using a composite regression interval mapping method via QTLExpress. A total of 18 QTL were detected, including 4 genome-wide significant QTL each for semen pH on pig chromosome (SSC) 2 and SSC12, for semen volume on SSC15, and for ejaculation times on SSC17. Fourteen suggestive QTL were found on SSC1, 2, 3, 4, 6, 9, 17 and 18. To our knowledge, this is the first report about the QTL for semen and ejaculation traits in pigs, providing a start point to decipher the genetic basis of these complex traits.     

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.     

Genetic diversity is related to climatic variation and vulnerability in threatened bull trout

Understanding how climatic variation influences ecological and evolutionary processes is crucial for informed conservation decision‐making. Nevertheless, few studies have measured how climatic variation influences genetic diversity within populations or how genetic diversity is distributed across space relative to future climatic stress. Here, we tested whether patterns of genetic diversity (allelic richness) were related to climatic variation and habitat features in 130 bull trout (Salvelinus confluentus) populations from 24 watersheds (i.e., ~4–7th order river subbasins) across the Columbia River Basin, USA. We then determined whether bull trout genetic diversity was related to climate vulnerability at the watershed scale, which we quantified on the basis of exposure to future climatic conditions (projected scenarios for the 2040s) and existing habitat complexity. We found a strong gradient in genetic diversity in bull trout populations across the Columbia River Basin, where populations located in the most upstream headwater areas had the greatest genetic diversity. After accounting for spatial patterns with linear mixed models, allelic richness in bull trout populations was positively related to habitat patch size and complexity, and negatively related to maximum summer temperature and the frequency of winter flooding. These relationships strongly suggest that climatic variation influences evolutionary processes in this threatened species and that genetic diversity will likely decrease due to future climate change. Vulnerability at a watershed scale was negatively correlated with average genetic diversity (r = ?0.77; P < 0.001); watersheds containing populations with lower average genetic diversity generally had the lowest habitat complexity, warmest stream temperatures, and greatest frequency of winter flooding. Together, these findings have important conservation implications for bull trout and other imperiled species. Genetic diversity is already depressed where climatic vulnerability is highest; it will likely erode further in the very places where diversity may be most needed for future persistence.     

Effects of dithiothreitol and boar on pronuclear formation and embryonic development following intracytoplasmic sperm injection in pigs

The objective of this study was to improve normal fertilization, male pronuclear formation and embryonic development following intracytoplasmic injection of dithiothreitol (DTT)-treated boar spermatozoa. To determine the effect of DTT treatment, frozen-thawed boar spermatozoa were treated with DTT for 0, 10, 30, and 60 min, and injected into porcine oocytes. The effects of DTT and male difference on normal fertilization and embryonic development were investigated. The mean normal fertilization rate in the groups treated with DTT for 30 min (73.8%) and 60 min (74.9%) was higher (P < 0.05) than that in the control group (49.3%). The mean blastocyst formation rate in the group treated with DTT for 30 min (23.2%) was higher (P < 0.05) than that in the other groups (8.7-10.9%). Among boars there was no difference in normal fertilization, but there was a significant difference between the non-treated and the DTT-treated groups. The mean rate of blastocyst formation was different (P < 0.05) among boars, and between the non-treated and DTT-treated groups. The mean number of cells in blastocysts was similar among the boars and between the non-treated and the DTT-treated groups. In conclusion, DTT treatment for 30 min increased the rate of normal fertilization and embryonic development to the blastocyst stage. Furthermore, the rate of blastocyst formation of oocytes injected with spermatozoa differed among boars.     

ICln is essential for cellular and early embryonic viability

pICln is a 26-kDa protein that is ubiquitously expressed and highly conserved from Xenopus laevis to Homo sapiens. The physiological functions of pICln remain to be established. To address this question, we disrupted the ICln gene in embryonic stem cells. We found that murine embryos lacking ICln die early in gestation (between stages E3.5 and E7.5). Furthermore, we found that ICln is essential for embryonic stem cell viability. Previously, we showed that pICln interacts directly with a homolog of a yeast protein that binds a PAK-like kinase and participates in the regulation of cell morphology and cell cycling. pICln also forms a complex with several core spliceosomal proteins, and this interaction may play a role in the regulation of spliceosomal biogenesis. Collectively, these data strongly suggest that pICln participates in critical cellular pathways, including regulation of the cell cycle and RNA processing.     

CUL-4A is critical for early embryonic development

Ubiquitin-mediated degradation targets cell cycle regulators for proteolysis. Much of the ubiquitin pathway's substrate specificity is conferred by E3 ubiquitin ligases, and cullins are core components of some E3s. CUL-4A encodes one of six mammalian cullins and is amplified and/or overexpressed in breast cancer, which suggests a role in regulating cell cycle progression. To examine CUL-4A's physiologic function, we generated a CUL-4A deletion mutation in mice. No viable CUL-4A(-/-) pups and no homozygous mutant embryos as early as 7.5 days postcoitum (dpc) were recovered. However, CUL-4A(-/-) blastocysts are viable, hatch, form an inner cell mass and trophectoderm, and implant (roughly 4.5 dpc), indicating that CUL-4A(-/-) embryos die between 4.5 and 7.5 dpc. Despite 87% similarity between the Cul-4A and Cul-4B cullins, the CUL-4A(-/-) lethal phenotype indicates that CUL-4A has one or more distinct function(s). Surprisingly, 44% fewer heterozygous pups were recovered than expected by Mendelian genetics, indicating that many heterozygous embryos also die during gestation due to haploinsufficiency. Taken together, our findings indicate that appropriate CUL-4A expression is critical for early embryonic development.     

Major morphological sperm abnormalities in the bull are related to sperm DNA damage

The influence of sperm morphology and chromatin integrity on bull fertility suggests a strong but undefined biological relationship between these two parameters. In this study we explore this relationship, making use of the Sperm Chromatin Dispersion test, which allows simultaneous observation of sperm abnormalities and DNA fragmentation. Based on spermatozoa from 17 Holstein-Friesian bulls, we determined a relationship between DNA fragmentation and the presence of the “so called” major-type sperm defects. Values for DNA fragmentation index (mean ± SEM) calculated from cells with major-type abnormalities were significantly (P < 0.05) higher (85.05 ± 5.00%) than those from abnormal forms classified as minor-type (17.89 ± 5.55%). Some of the sperm abnormalities, such as double forms, narrow base heads, small heads, shortened tails and proximal cytoplasmic droplets, were only associated with sperm showing fragmented DNA. The simultaneous assessment of sperm morphology and DNA fragmentation has the potential to improve the efficacy of sperm quality assessment in this species.     

The p24 family member p23 is required for early embryonic development

The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.     

Trout sperm motility. The transient movement of trout sperm is related to changes in the concentration of ATP following the activation of the flagellar movement

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours. We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20-40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20-30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not stop abruptly, flagella kept beating and stopped only after 1-2 min. When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter. The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest. In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due to a low mitochondrial oxidative phosphorylation capacity.     

Primary productivity is weakly related to floristic alpha and beta diversity across Australia

Aim Ecosystem functions such as productivity may be influenced not only by the biological diversity at each location (α‐diversity) but also by the biological turnover between locations (β‐diversity). We perform a continental‐scale test of the strength and direction of relationships between gross primary productivity (GPP) and both α‐ and β‐diversity. Location Continental Australia. Methods Species occurrence records were used to quantify the taxonomic α‐diversity of vascular plants in approximately 11,000 1 km × 1 km grid cells across Australia, and to calculate the average β‐diversity within a 10‐km radius around each cell. The magnitude and variability of monthly, MODIS‐derived remotely sensed GPP (2001–12) were summarized for continental Australia, as were rainfall and temperature over the same period. Generalized additive models were then used to test whether the magnitude or variability of GPP were distinctly influenced by either biodiversity measure, over and above the influence of environmental conditions. Results Precipitation and temperature explained large proportions of deviance in the magnitude (75.6%) and variability (38.3%) of GPP across the Australian continent. GPP was marginally more strongly related to species richness than it was to species turnover. However, neither diversity measure provided substantial increases in the explanatory power of GPP models over and above that of environment‐only models (always < 1%). Main conclusions The relationship between primary productivity and taxonomic α‐ and β‐diversity was weak for the Australian flora. Our findings question the generality of key assumptions, predictions and results in the literature regarding the strength of empirical relationships between productivity and biodiversity across multiple biological levels (α‐, β‐ and γ‐diversity) at macroecological scales.