Blood typing South American camelids

Preliminary blood typing tests were made on New World camelids, guanacos, llamas, and two hybrids. Erythrocyte samples were tested against a battery of cattle blood typing reagents. Three different reagents were prepared from rabbit anti-erythrocyte sera. Transferrin variation and lectin polymorphism also were observed. No naturally occurring isoantibodies were found. Blood typing tests of New World camelids were shown to be feasible for studies of taxonomic relationships.     

Eight polymorphic microsatellite markers for Rhinanthus minor

Little research has been carried out on the genetics of Rhinanthus minor to date. To enable study of this species, eight polymorphic microsatellite loci were developed, using a genomic library enriched for microsatellites. All loci are polymorphic in the two UK populations tested, Bardister and Oxwich Bay. These microsatellite markers will be useful for studying genetic structure and subspecies differences of R. minor.     

Embryo transfer in domestic South American camelids

Intraspecific and interspecific embryo transfer in domestic South American camelids is developing into a well-established technique. Reports reveal many benefits of using reproductive biotechnologies to allow rapid propagation of alpacas and llamas of high genetic merit (e.g., high fiber quality, preserve color variation). The objective of this review is to provide up-to-date information about embryo transfer in domestic South American camelids. Specific information is provided on criteria for male selection, donor and recipient synchronization, the practice of single- vs. super-ovulation protocols, embryo recovery and transfer techniques, advances in cryopreservation of embryos, results of intra- and inter-specific transfer, and the future of the embryo transfer in domestic South American camelids.     

Induction of superovulation in South American camelids

The development of assisted reproductive technologies such as embryo transfer (ET), artificial insemination (AI) and in vitro fertilization (IVF) in South American camelids is considerably behind that of other livestock species. Poor success of the embryo transfer technique has been related to a lack of an effective superstimulatory treatment, low embryo recovery rate, and the recovery of hatched blastocysts that are not conducive to the cryopreservation process. Superstimulation has been attempted using equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) during the luteal, or the sexually receptive phase, sometimes given at follicular wave emergence. The rationale for inducing a luteal phase prior to or during superstimulation in camelids is not clearly understood, but it may simply reflect an empirical bias to conventional methods used in other ruminants. The number of ovulations or CL varies widely among studies, ranging from 2 to more than 15 per animal, with the number of transferable embryos ranging from 0 to 4 per animal. The control of follicular growth combined with superstimulatory protocols has resulted in a more consistent ovarian response and a greater number of follicles available for aspiration and oocyte collection. Recent studies in llamas have demonstrated that the use of ovulation inducing treatments or follicle ablation can synchronize follicular wave emergence allowing the initiation of gonadotropin treatment in the absence of a dominant follicle resulting in a more consistent ovulatory response. Few studies in alpacas have been reported, but it appears from recent field studies that the ovarian response is more variable and that there is a greater number of poor responders than in llamas. A review of superstimulation protocols that have been used in llamas and alpacas in the last 15 years is provided, including a discussion of the potential of protocols designed to initiate treatment at specific stages of follicular growth.     

A review on reproduction in South American camelids

In this paper, aspects of reproductive physiology and endocrinology, as well as sexual behaviour in South American camelids are reviewed. Because of the many unique features of reproduction in these animals, the application of advanced breeding techniques that are routinely used in other domestic species has been slow and, in some cases, are not applicable. Relatively high embryonic loss and the capacity to carry only one offspring at a time limit production in females. Furthermore, some 20% of females do not conceive following mating. Research is needed to elucidate the causes of embryonic loss, particularly in relation to the preferential location of embryos in the left uterine horn and the apparent differential luteolytic activity of the two uterine horns. A fuller understanding of the endocrine changes and mechanisms accompanying folliculogenesis, estrus, induction of ovulation and luteal regression may led to treatments that provide better control of ovulation and enhance the quality and viability of eggs shed. In the male, the hormonal interactions involved in facilitating libido need to be established and the underlying causes of declining libido resulting from continued exposure to estrous females, identified. More importantly, there is a need to develop a routine method to collect semen from animals on farm, or in studs. Deficiencies in this area have long hampered the evaluation of sires through proper assessment of their semen quality and in the application of artificial insemination. In addition, establishment of techniques to freeze semen from these species has obvious advantages for breeding, including crossbreeding between species to improve products such as fibre and to assist in preservation of some of the more endangered New World camelids.     

Semen preservation and artificial insemination in domesticated South American camelids

Semen preservation and artificial insemination in South American camelids are reviewed giving emphasis to work done in Peru and by the authors. Reports on semen evaluation and the preservation process indicate that semen of alpacas and llamas can be manipulated by making it liquid first. Collagenase appears to be the best enzyme to eliminate viscosity. Tris buffer solution maintains a higher motility than egg-yolk citrate, phosphate buffered saline (PBS), Triladyl, and Merck-I extenders. Cooling of semen took 1 h after collected, and equilibrated with 7% glycerol presented a better motility and spermatozoa survival at 1, 7, 15 and 30 days after being slowly frozen in 0.25 mL plastic straws. Trials of artificial insemination with freshly diluted semen and frozen–thawed semen are encouraging and needs to be tested extensively under field conditions. Recently, fertility rates varied from 3 to 67%. Semen preservation and most important, artificial insemination appear to be a reality, and could be used to improve the genetic quality of alpacas and llamas.     

Artificial insemination in South American camelids and wild equids

An overview of the present status of the use of artificial insemination (AI) in South American camelids and wild equids is offered. Technical aspects of semen collection, dilution and cryopreservation have limited the development and use of AI in camelid and equid species. To-date, efficiency is low but progress has been made and viable offspring have been produced through the use of AI in domestic South American camelids using both fresh and frozen semen. The origin, composition, and function of the viscous component of camelid seminal plasma remain a mystery and an obvious area for future research. A better understanding of the normal constituents of seminal plasma will enable the rational design of semen extenders suitable for camelids. Post-thaw sperm viability is very low, and studies are needed to address questions of optimal freezing and thawing procedures as well as the insemination dose. The basis for differences in reported pregnancy rates with sexed and frozen semen in domestic equids, and the ultimate success of AI in wild equids will require continued research into the "stallion effect", extenders and cryoprotectants, optimal volume and number of spermatozoa, temperatures during handling, processing an transport, and insemination techniques. In both camelids and equids, research on domestic species under controlled conditions provides and excellent opportunity to develop effective semen handling techniques for application in wild and endangered species of the respective families.     

Eight new polymorphic microsatellite DNA markers for Sakhalin taimen Parahucho perryi

We developed eight polymorphic microsatellite markers for the endangered salmonid Parahucho perryi from genomic libraries enriched for (GACA)n and (GATA)n repeat sequences. Emphasis was placed on developing highly polymorphic, perfect repeats that could be scored with confidence. Six tetra- and two di-nucleotide loci were screened in 49 individuals from two populations on Sakhalin Island, Russia. Allelic variation was high with eight to 23 alleles per locus and expected heterozygosity ranging from 0.48 to 0.93. These highly variable markers should prove useful in evaluating inbreeding, gene flow and population structure in Sakhalin taimen throughout its range.     

Eight microsatellite markers for the San Esteban chuckwalla,Sauromalus varius

We characterize eight polymorphic microsatellite loci for the San Esteban chuckwalla, Sauromalus varius, which are being used as markers in a study of the genetic results from a decade of captive breeding in this endangered species. Observed heterozygosity estimates for S. varius ranged from 0.00 to 0.30, whereas expected heterozygosity estimates ranged from 0.06 to 0.58.     

Development and characterization of microsatellite markers for the wild South American Passiflora cincinnata (Passifloraceae)

? Premise of the study: We developed the first set of microsatellite markers for the passion fruit, Passiflora cincinnata, to provide tools for further study of its genetic diversity and to support current conservation and genetic studies. ? Methods and Results: We used a microsatellite-enriched library approach to isolate and characterize 25 new molecular markers. Seven primer pairs successfully amplified polymorphic microsatellite regions, with an average of five alleles per locus. The mean values of expected and observed levels of heterozygosity were 0.516 and 0.525, respectively. ? Conclusions: The microsatellite markers identified in this study may be valuable tools for population genetic studies, and this set of markers also may be useful in the design of a genetic conservation strategy, mating system, and hybridization studies.     

In vitro production of embryos in South American camelids

Studies in reproductive biotechnology techniques have been minimal in South American camelids (SAC). Complex reproductive characteristics of these species contribute to slow progress. Nevertheless, some techniques, such as in vitro fertilization, intracytoplasmic sperm injection and nuclear transfer have been applied and have produced advances in knowledge on embryo environment and in vitro conditions necessary for development. Embryo production may have a high impact in both domestic and wild camelids population. Studies addressed to improve in vitro embryo production and oocyte collection could be a potential key to develop IVF and embryo production as a routine procedure in camelids.     

Eight novel microsatellite DNA markers in Rhodiola rosea L.

An enrichment procedure was used to develop eight di- and tri-microsatellites for Rhodiola rosea L., whose root extract is used as a powerful adaptogen. In order to evaluate the polymorphism and allelic variation at each locus, 48 individuals sampled in two different locations in Trentino region (Italy) were analyzed. All loci were polymorphic, showing 2–5 alleles per locus and an observed heterozygosity ranging from 0.09 to 0.76. HWE test shows significant deviation for five loci in two different populations. No linkage disequilibrium among loci was observed. These SSR markers are being used for the study of the genetic variation within and among populations in the Trentino Alps.     

Eight polymorphic microsatellite markers for the spotted babylon, Babylonia areolata (Buccinidae)

The spotted babylon, Babylonia areolata, is one of the most extensively cultured marine mollusks in southeast Asia. Eight polymorphic microsatellite markers were developed for this species, from a microsatellite-enriched library. These markers, characterized in 32 individuals from a hatchery population, were polymorphic, with allele numbers ranging from 6 to 18 per locus, expected and observed heterozygosities ranging from 0.68 to 0.94 and 0.56 to 0.81, respectively. One locus (HUBA09) showed significant deviation from Hardy-Weinberg equilibrium, probably due to the presence of null alleles. These microsatellite loci should be useful for future population genetic studies and marker-assisted breeding in this species.     

Evaluation of insulin resistance in two kinds of South American camelids: llamas and alpacas

Insulin resistance was evaluated in South American camelids, llamas and alpacas, by use of the minimal model test and the insulin tolerance test. Animals were catheterized for long-term studies and tamed to minimize stress during evaluation. Results indicated a low insulin sensitivity index (SI) = 0 to 0.97, median = 0.39 x 10(-4) min/uIU x ml, about a fifth the value in other mammals and humans. The KITT was between 1.43 and 3.19 %/min, also significantly lower than that reported for humans. Glycosylated hemoglobin concentration was 6%, and HbAlc concentration was 5.5%; red blood cell lifetime, as measured by use of the 51Cr method, was 120 days, similar to the value in humans. We concluded that llamas and alpacas have naturally higher blood glucose concentration than do humans and other mammals during the glucose tolerance test. Using the same mathematical tools to evaluate glucose metabolism as those used in people, South American camelids appear to be resistant to insulin. Thus, the South American camelid may be a useful new animal model for the study of sugar metabolism and various facets of diabetes mellitus, especially protection from the deleterious effects of glycosylation.     

Eight polymorphic microsatellite markers in the Andalusian belladonna (Atropa baetica, Solanaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for the critically endangered Atropa baetica. These are the first microsatellite loci reported for Atropa species. The total number of alleles found was 18, the expected heterozygosity ranged from 0.198 to 0.505. These markers will be useful to establish the real census of individuals and the genetic diversity both within and among the different populations of A. baetica.     

Ag-NOR staining and in situ hybridization of rDNA in the chromosomes of the South American camelids

The location and frequency of Ag-stained NORs and sites of rDNA hybridization were studied in the chromosomes of the South American camelids. In the four camelids these regions occur distally on chromosomes 18, 21, and 27 and the smallest biarmed elements. Quantitative analysis of NOR distribution showed variations between both cells and species. In llama, guanaco and alpaca the NORs number averaged 6 per cell, this being higher than in vicuña where the average was 3. Relative frequencies of NOR-bearing chromosomes in the four camelids were similar. Yet, in vicuña the virtual absence of NOR sites on one of the smallest biarmed pairs was observed. The rDNA sites assessed in llama and vicuña by in situ hybridization with cloned 18S DNA were coincident with the NOR locations and with the frequencies characteristics for each species. Moreover, varying the exposure time of the autoradiographs, labeling patterns specific for each camelid were observed. Grain counts on individual chromosomes indicated that under our conditions one month exposure is enough to demonstrate all the rDNA sites available in the complement of llama. Conversely, at least two months are necessary to show the total sites existing in vicuña. Most probably this finding reflects the presence of variations in the amount of copies of the ribosomal genes per chromosome.     

Eight polymorphic microsatellite loci markers for the barnacle Balanus amphitrite (syn. Amphibalanus amphitrite) Darwin 1854

Balanus amphitrite is a widespread species of barnacle. It is frequently studied, and of great importance to the marine coatings industry due to its significant abundance as a fouling organism on commercial shipping. We isolated and characterized eight highly polymorphic microsatellite loci, to aid in the determination of population genetic structure within this species. All loci showed considerable genetic variation with the number of alleles ranging from two to 14. Expected heterozygosity ranged from 0.74 to 0.98.     

DNA composition in South American camelids I. Characterization and in situ hybridization of satellite DNA fractions

The DNA composition and the in situ hybridization of satellite fractions were analysed in the New World camelids llama, alpaca, guanaco and vicuña. In the four camelid forms, it was possible to identify a similar main band DNA and five satellite fractions (I–V) with G+C base contents ranging from 32% to 66%. Satellites II–V from llama were in situ reannealed on chromosomes from the four camelid forms. The results obtained were: (a) the four satellites hybridized with regions of C-banding (centromeric regions of all chromosomes and short arms of some autosomes); (b) in general, homologous hybridizations (llama DNA versus llama chromosomes) were more efficient than heterologous reassociations; there were however three exceptions to this rule (vicuña and alpaca satellite fraction II, chromosome group B; vicuña fraction V, chromosome groups A and B); (c) X chromosomes from the four camelids had satellites III–V but lacked satellite II, (d) no satellite fraction was detected on chromosome Y. The analysis of the in situ hybridization patterns allowed to conclude that most or all C-banded chromosome regions comprise several satellite DNA fractions. It is, moreover, proposed that there is an ample interspecies variation in the number of chromosomes that cross-react with a given satellite fraction. Our data give further support to the close genomic kinship of New World camelids.