Assessing the diversity and abundances of larvae and juveniles of coral reef fish: a synthesis of six sampling techniques

Due to an increasing emphasis for fish population survey and regulation, efficient tools for evaluating the abundance and diversity of fish from various life stages are needed, especially for coral reef species that present a high taxonomic diversity. The characteristics of six different techniques used for sampling pelagic larvae (a plankton-net and two light-traps), newly settled juveniles (one type of artificial reef), and older juveniles (an underwater seine net in seagrass and macroalgal beds, and rotenone poisoning in coral patches) are described in this study. Larvae belonging to 70 families and juveniles belonging to 34 families were collected. An analysis of similarity (ANOSIM) showed that the taxonomic composition of assemblages collected with the plankton-net and the two light-traps were overlapping but clearly different, due to the higher occurrence of Gobiidae in the plankton-net and of Pomacentridae in both light-traps. Larvae being 2–4 mm standard length (SL) dominated in the plankton-net, whereas larvae being 9–11 mm SL dominated in both light-traps. Pomacentridae juveniles were more abundant in rotenone samples, whereas Labridae dominated in the underwater seine. Juvenile fish collected with the artificial reefs, the underwater seine, and rotenone poisoning largely overlapped in size, with mean sizes of 22, 38, and 33 mm SL, respectively. Seven families were caught by the six sampling techniques, but with unequal success. This study provides ecologists and managers with a unique review of six techniques for sampling a wide range of developmental stages of young fish in different habitats of a coral reef lagoon.

Superovulation and embryo transfer in Holstein cattle using sexed sperm

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 × 10⁶ sexed (X-sorted; n = 5) or unsexed (n = 5) frozen-thawed sperm from one bull at 12 and 24 h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P < 0.0001). In Experiment 2, donors were inseminated twice with 5 × 10⁶ sexed unfrozen sperm (n = 10) or sexed frozen-thawed sperm (n = 9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P < 0.05) inseminated twice with 5 × 10⁶ sperm/dose (10 × 10⁶ total). We concluded that the use of sexed frozen-thawed sperm (≥90% X-sperm biased and 10 × 10⁶ total sperm) may be economically viable for commercial MOET programs in Holstein heifers.     

Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species

Approximately 503 of the known species of birds are classified as ‘endangered’ or ‘critical’. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific.     

Effect of natural or vaccine-induced porcine circovirus type 2 (PCV2) immunity on fetal infection after artificial insemination with PCV2 spiked semen

The objectives of this study were to determine if vaccination against porcine circovirus type 2 (PCV2) or previous PCV2 infection of the dam are sufficient to prevent fetal infection when dams are artificially inseminated with PCV2-spiked semen. Nine sows (Sus domestica) were allocated into three groups of three dams each: The PCV2 naïve negative control Group 1 was artificially inseminated with extended PCV2 DNA negative semen during estrus, whereas the extended semen used in the vaccinated Group 2 (PCV2 vaccine was given 8 wk before insemination) and PCV2-exposed Group 3 (infected with PCV2 12 wk before insemination) was spiked with 5 mL of PCV2 inoculum with a titer of 10^4.2 tissue culture infectious dose (TCID₅₀) per milliliter at each breeding. The dams in the vaccinated and PCV2-exposed groups were positive for PCV2 antibody but negative for PCV2 DNA in serum at the time of insemination. Three negative control dams, two vaccinated dams, and three dams with previous PCV2 exposure became pregnant and maintained pregnancy to term. After artificial insemination, viremia was detected in one of three vaccinated dams and in two of three dams with previous PCV2 exposure. At farrowing, PCV2 infection was not detected in any piglets or fetuses expelled from the negative control dams or from dams with previous PCV2 exposure. In litters of the vaccinated dams, 15 of 24 live-born piglets were PCV2 viremic at birth, with 6 of 26 fetuses having detectable PCV2 antigen in tissues. In conclusion, vaccine-induced immunity did not prevent fetal infection in this sow model using semen spiked with PCV2.     

Artificial insemination for the propagation of CANDES: the reality!

Conservation is about protecting and nurturing species so that they can survive, not only now, but also into the future. Ideally this means protecting genetically diverse populations and not simply breeding a few individuals. Unfortunately, this point is often overlooked by reproductive technologists, especially if they are more accustomed to working with humans, companion animals or agricultural species, where the goals are more usually directed towards obtaining offspring from particular individuals. This approach has tended to antagonise the conservation community, who are quick to develop an unreasonable suspicion of technological solutions, partly because they are unfamiliar with the scientific principles that underpin the reproductive technology. Unfortunately, this mutual failure to recognise that all parties are actually well meaning, has led to separate cultures that barely communicate with each other and thus fail to capitalise on the potential benefits that would come from a good working relationship. Notable successes with reproductive technology have only emerged where such relationships have been forged. In this review, we highlight, mainly for the benefit of the technologist community, the need to foster good working relationships with conservation managers and to recognise that the latest hi-tech approach to animal breeding is more likely to engender suspicion than enthusiasm.     

NetMHCpan,a method for MHC class I binding prediction beyond humans

Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct MHC molecule. The potentially unique specificity of the majority of HLA alleles that have been identified to date remains uncharacterized. Likewise, only a limited number of chimpanzee and rhesus macaque MHC class I molecules have been characterized experimentally. Here, we present NetMHCpan-2.0, a method that generates quantitative predictions of the affinity of any peptide–MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide immunologists in interpreting cellular immune responses in large out-bred populations is demonstrated. Further, we used NetMHCpan-2.0 to predict potential binding peptides for the pig MHC class I molecule SLA-1*0401. Ninety-three percent of the predicted peptides were demonstrated to bind stronger than 500 nM. The high performance of NetMHCpan-2.0 for non-human primates documents the method’s ability to provide broad allelic coverage also beyond human MHC molecules. The method is available at . Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.     

Bidirectional selection for female propensity to remate in the bean beetle, <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis>

The evolution of female multiple mating, or polyandry, is difficult to comprehend and thus has been the subject of a large number of studies. However, there is only a little evidence for genetic variation in polyandry, although the evolution of a trait via selection requires genetic variation that enables the trait to respond to selection. We carried out artificial selection for increased and decreased female propensity to remate as a measure of polyandry to investigate whether this trait has a genetic component that can respond to selection in the adzuki bean beetle, Callosobruchus chinensis. Artificial selection produced responses in both directions and divergence between the selection lines in the female propensity to remate. Although the experimental design adopted in this study selected jointly for female receptivity to remating, which is a trait of females, and male ability to inhibit female remating—both of which are associated with female propensity to remate—the observed response to selection was attributable only to the female receptivity to remating. This study indicates that the female receptivity to remating has significant additive genetic variation and can evolve according to whether remating is advantageous or disadvantageous to females in C. chinensis.     

Transglutaminases expression in human supraspinatus tendon ruptures and in mouse tendons

The ethiopathogenesis of rotator cuff disease remains poorly understood. Many studies advocate the importance of extra cellular matrix for the homeostasis of connective tissue. Transglutaminase enzymes family has been studied in the context of connective tissue formation and stabilisation. Here, we investigated transglutaminases expression pattern in biopsies of normal and injured supraspinatus tendons of human shoulders and in the Achilles tendons of transglutaminase 2 knock-out and wild-type mice. Our results show that different transglutaminase family members are differentially expressed in human and mouse tendons, and that transglutaminase 2 is down-regulated at mRNA and protein levels upon human supraspinatus tendon ruptures.     

Effect of simulated macroalgae on the fish assemblage associated with a temperate reef system

Increased habitat complexity is supposed to promote increased diversity, abundance and biomass. This study tested the effect of the macroalgal cover on temperate reef fishes by mimicking macroalgae on artificial reefs in NW Sicily (Mediterranean Sea). Macroalgal cover affected reef fishes in different ways and independently of intrinsic temporal trends. The fish assemblages of manipulated and control artificial reef units differed in the relative abundances of the associated species, but little in species composition. In line with studies in seagrass habitats, fishes were most abundant in reefs covered by artificial macroalgae. Three species (Boops boops, Serranus scriba and Symphodus ocellatus) exhibited consistently greater abundance on vegetated reef units than on control reef units. The total number of species and the abundance of three particular species (S. scriba, S. ocellatus and Thalassoma pavo) displayed temporal trends which were independent on short and large temporal scales. Only fish total biomass and one species (Spicara flexuosa) displayed strong effects of interaction among the experimental factors. Mechanisms to explain these findings are discussed from observational evidence on habitat use and interactions among multiple species. This study highlights that manipulative experiments involving repeated sampling of fish in artificial habitats appear to be a valid approach to study fish-habitat relationships in fluctuating environments. It is also concluded that macroalgae mimics may serve as a tool for restoring lost marine vegetated habitats when current human-induced conditions prevent the recovery of pristine macroalgal stands.