Effect of Three Semen Extenders on Sperm Quality and In Vitro Fertilization Rates of Fresh and Cryopreserved Sperm Collected from Llama (Lama glama) Vas Deferens.

The advances in Assisted Reproductive Technologies (ARTs) applied in South American camelid species are still scarce. The aim of this study was to compare the effects of three semen extenders, before and after the cryopreservation of spermatozoa obtained from the vas deferens, on sperm quality parameters and in vitro fertilization rates of llama (Lama glama) oocytes. Mature fertile llama males (Lama glama; n = 6; age: 48-60 mo.; BCS: ~2.7) were included in the study. Sperm samples were collected from each male using the surgical technique of the vas deferens deviation. Then, the sperm samples were pooled and diluted with the Tris-EY, Andromed®, or BioxCell® extender in order to subsequently carry out the sperm cryopreservation process. The sperm quality assessment related to each extender was performed before and after cryopreservation with regard to sperm morphological abnormalities, acrosome integrity, sperm viability, membrane permeability, and sperm motility traits. Moreover, in vitro fertilization (IVF) procedures were carried out to evaluate the in vitro fertility of the cryopreserved sperm samples using each extender. Overall, significant differences were observed before and after cryopreservation regarding acrosome integrity, sperm viability, membrane permeability, and sperm motility traits among the extenders used, where Tris-EY and Andromed® were better than BioxCell® (p < 0.05); however, no differences were observed regarding the sperm morphological abnormalities among extenders (p > 0.05). Moreover, multiple differences were observed with regard to the velocity and linearity kinematic parameters obtained by computerized analysis before and after the cryopreservation process, irrespective of the extender used (p < 0.05). Finally, differences were observed regarding the in vitro fertilization rates among the different extender-derived samples (p < 0.05). In conclusion, the sperm quality using Tris-EY and Andromed® was better before and after cryopreservation compared to that using BioxCell®. Although the number of fertilized oocytes obtained after the IVF process between Tris-EY and Andromed® was similar, Andromed®-derived samples showed the best sperm quality results before and after cryopreservation. This indicates that the cryopreservation extender is a determining factor in significantly improving in vitro fertilization rates when using sperm samples obtained from vas deferens in llama (Lama glama) males.

Prevalence and molecular characterization of Sarcoptes scabiei from vicuñas (Vicugna vicugna) from Southern Peruvian Andes.

Sarcoptic mange is a disease caused by an infectious parasite in the vicuñas (Vicugna vicugna) from South America. Although molecular studies have provided much information about the epidemiology of this disease, this information is still unknown in vicuñas. This study determined the prevalence and molecular characterization of Sarcoptes scabiei from vicuñas from Southern Peruvian Andes. During the 2018 shearing season, 181 vicuñas were clinically evaluated for lesions compatible with mange. Sarcoptes scabiei was detected in 35 (19.3%) vicuñas, and 50 mites from 25 vicuñas were selected for molecular analyses of the mitochondrial (cox1) and nuclear (ITS2) genetic markers. Molecular analyses of the cox1 and ITS2 sequences showed an identity of 94­99% and 99.8­100% with previous S. scabiei sequences registered in the GenBank, respectively. Sequence polymorphisms were more evident in the ITS2 than in the cox1, but only the cox1 had an association with the host. Phylogenetic analysis of S. scabiei cox1 sequences from vicuñas showed a cluster with S. scabiei cox1 sequences from canids, suggesting that the origin of S. scabiei from vicuña is associated with canid mites. This research is the first molecular analysis of S. scabiei from vicuñas. Future molecular studies will be necessary to determine the species variety, geographic segregation and host­parasite adaptation for this vicuña's mite.

Soy lecithin as a potential alternative to powdered egg yolk for buck sperm cryopreservation does not protect them from mitochondrial damage.

The aim of this study was to address whether soy lecithin (SL) was an effective non-penetrating cryoprotectant for buck sperm cryopreservation in the presence of seminal plasma. There was also an attempt to determine the optimal concentration of BHT as an antioxidant in powdered egg yolk (PEY) or in SL based media. Two ejaculates were collected from six bucks and mixed ejaculates were aliquoted into washed, using centrifugation procedures, and unwashed samples. In Experiment 1, washed sperm were re-suspended in PEY (15%) or SL (1%) media, while unwashed semen was only diluted in SL medium. In Experiment 2, washed and unwashed sperm were diluted in PEY and SL media, respectively, with there being different BHT concentrations (0.6, 2.0 and 5.0 mM). In both experiments, after 4 h of refrigeration, there were no differences neither in sperm viability nor plasma membrane functional integrity (HOST) between groups when there were evaluations using eosin-nigrosine staining. After thawing, however, there was a negative effect on motility of washed sperm preserved in SL media. Furthermore, results from cytometry evaluations indicated there was a larger population of thawed sperm with intact plasma (SYBR-14+/PI-) and acrosome (PE-PNA-) membranes, but inactive mitochondria (Mitotracker deep red-) when SL media were used. When there was BHT supplementation, there was only a slight enhancement of motility of spermatozoa preserved in PEY media with 5 mM BHT. In conclusion, when effectiveness and efficiencies are considered, PEY is the non-penetrating cryoprotectant that should be utilized for buck sperm cryopreservation.

Effect of donkey seminal plasma on sperm movement and sperm-polymorphonuclear neutrophils attachment in vitro.

To evaluate the effect of seminal plasma in endometrial inflammation in donkeys, samples from fresh pure, fresh diluted and frozen-thawed semen of three different jackasses were co-incubated in water bath at 37°C with uterine Jennie's secretions collected 6h after artificial insemination with frozen-thawed donkey semen. Individual sperm movement parameters using the computerised sperm analysis system (CASA) and sperm-polymorphonuclear neutrophils (sperm-PMN) attachment observed in Diff-Quick stained smears were evaluated at 0, 1, 2, 3 and 4h of co-incubation. Controls consisted of incubating diluted or frozen-thawed sperm in the absence of uterine secretions. For data analyses, a repeated measures ANOVA was performed with incubation time as intra-subject factor and with treatment and donkey as inter-subject factor, followed by a post-hoc Bonferroni's test. Greater values (P<0.05) of sperm-PMN percentages and a loss of progressive motility were observed in frozen-thawed semen compared with pure and diluted fresh semen samples throughout the incubation time. In addition, the presence of seminal plasma in fresh and diluted semen samples reduced the inflammatory response of polymorphonuclear neutrophils produced after insemination by suppressing the sperm-PMN attachment in vitro. Motility sperm parameters analysed by CASA were also less affected than those in frozen-thawed semen samples. In conclusion, seminal plasma in jennies appears to have a modulation on the endometrial response after artificial insemination with frozen-thawed donkey semen. As a result, spermatozoa with the greater motility characteristics are selected.