Sarcocystis spp. of New and Old World Camelids: Ancient Origin, Present Challenges.

Sarcocystis spp. are coccidian protozoans belonging to the Apicomplexa phylum. As with other members of this phylum, they are obligate intracellular parasites with complex cellular machinery for the invasion of host cells. Sarcocystis spp. display dixenous life cycles, involving a predator and a prey as definitive and intermediate hosts, respectively. Specifically, these parasites develop sarcocysts in the tissues of their intermediate hosts, ranging in size from microscopic to visible to the naked eye, depending on the species. When definitive hosts consume sarcocysts, infective forms are produced in the digestive system and discharged into the environment via feces. Consumption of oocyst-contaminated water and pasture by the intermediate host completes the parasitic cycle. More than 200 Sarcocystis spp. have been described to infect wildlife, domestic animals, and humans, some of which are of economic or public health importance. Interestingly, Old World camelids (dromedary, domestic Bactrian camel, and wild Bactrian camel) and New World or South American camelids (llama, alpaca, guanaco, and vicuña) can each be infected by two different Sarcocystis spp: Old World camelids by S. cameli (producing micro- and macroscopic cysts) and S. ippeni (microscopic cysts); and South American camelids by S. aucheniae (macroscopic cysts) and S. masoni (microscopic cysts). Large numbers of Old and New World camelids are bred for meat production, but the finding of macroscopic sarcocysts in carcasses significantly hampers meat commercialization. This review tries to compile the information that is currently accessible regarding the biology, epidemiology, phylogeny, and diagnosis of Sarcocystis spp. that infect Old and New World camelids. In addition, knowledge gaps will be identified to encourage research that will lead to the control of these parasites.

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase.

The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal-Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p < .05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p < .05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p < .05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.

New protocol to separate llama sperm without enzymatic treatment using Androcoll-E™.

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p Ëƒ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies.

Evaluation of the cryoprotective effect of seminal plasma on llama (Lama glama) spermatozoa.

In South American camelids, sperm survival is low after thawing and poor results are obtained when artificial insemination is performed with cryopreserved semen. The aim of this study was to evaluate the effect of different percentages (10% and 50%) of seminal plasma added prior to the process of cryopreservation and also to evaluate the absence of seminal plasma on llama sperm survival after freezing and thawing. A total of 15 ejaculates from five adult llama males (n = 5; r = 3) were evaluated. A significant decrease in sperm motility, viability, membrane function and intact acrosomes was observed in thawed samples (0%, 10% and 50%) when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > 0.05), but a significant increase (p < 0.05) in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples compared to raw semen. Higher percentages of total and progressive sperm motility were observed when 0% and 10% of seminal plasma were used compared to 50%. However, no statistical differences were established for sperm viability, membrane function, morphology, acrosome status and DNA quality between thawed treatments. To conclude, neither of the percentages of seminal plasma used showed superiority nor cryoprotective effect on llama sperm survival.

Production, Preservation, and Transfer of South American Camelid Embryos.

The current review summarizes progress in the field of in vitro and in vivo production of South American Camelid embryos. Both methods require ovarian superstimulation (with FSH and eCG) to obtain multiple ovulations (in vivo embryo production) or to induce follicle growth for oocyte collection (in vitro embryo production). Moreover, superstimulation entails prior administration of hormones that inhibit follicular growth (progesterone, progestagens, and estrogens). Cumulus-oocyte complexes obtained must mature in vivo (buserelin administration) or in vitro to then be subjected to in vitro fertilization or intracytoplasmic sperm injection. All these techniques also require morphologically normal, motile spermatozoa to achieve fertilization. Methods used to decrease semen viscosity and to select the best spermatozoa (Percoll®; Androcoll-ETM) are described. Additionally, nuclear transfer or cloning has been applied in llamas. Up to now, embryo deep-freezing and vitrification have progressed slowly but are at the height of development. Embryos that are obtained by any of these techniques, either in vivo or in vitro, need to be transferred to synchronized recipient females. The best results are achieved after transfer to the left uterine horn with an ipsilateral ovulation. No live offspring have been obtained after the transfer of cryopreserved embryos. Applying reproductive biotechnologies, such as those described, will permit the expansion of genetically selected animals in the population and also that of wild camelid species, vicunas, and guanacos, whose embryos could then be transferred to the uterus of domestic species.

In vitro production of llama (Lama glama) embryos by IVF and ICSI with fresh semen.

The interest for South American camelids has increased in the last years. The aim of the present research was to compare the in vitro production of Lama glama embryos using two techniques: in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). For IVF technique, we compared the effect of adding or not, heparin, penicillamine and hypotaurine as sperm capacitating agents. In the oocyte group subjected to ICSI, activation with or without, ionomycin and 6-dimethylaminopurine (6-DMAP) was assessed. Semen samples were obtained by electroejaculation and incubated at 38 degrees C in a 25% (v/v) collagenase solution. The cleavage and embryo development rates were compared between the different experimental groups. Only the number of cleaved oocytes was less when ICSI with no activation was used (p<0.05).