Use of commercial extenders, with and without the addition of egg yolk, for cooling llama semen.

The objective of this study was to evaluate the effect of two commercial extenders, AndroMed® (AM) and Androstar® Plus (AS) both with and without the addition of egg-yolk (EY), for cooling llama semen. A total of sixteen ejaculates were collected from four males. Each ejaculate was divided into four aliquots and diluted with: AM, AM with 20 % EY (AM-EY), AS and AS with 20 % EY (AS-EY) and then cooled to 5 °C in an Equitainer®. Evaluations were carried out in raw semen, after dilution (0 h) and after 24 and 48 h of cooling. Data were analysed using either Friedman or ANOVA. Although total motility decreased in all cooled samples compared to the corresponding 0 h (P < 0.05), the highest percentages were observed in AM-EY being significantly higher than all other cooled samples after 24 h and higher than AS and AS-EY after 48 h. No significant differences were observed in the percentages of live acrosome-intact sperm between extenders at all times tested. A significant decrease in the percentage of sperm membrane osmotic function was observed in samples cooled with AS and AS-EY after 24 and 48 h vs. raw semen and in AM 48 h vs. raw semen. Finally, a significant increase in the percentage of sperm with abnormal tails was observed in the samples cooled with AS and AS-EY. Of all the extenders used, AndroMed® could be considered an option for cooling llama semen and the addition of EY to this extender improves its effectiveness. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Cryopreservation of llama semen using a combination of permeable cryoprotectants.

Semen cryopreservation is not available for massive use in South American Camelids (SACs) due to the lack of an efficient protocol and the low pregnancy rates obtained with artificial insemination (AI). The use of a single cryoprotectant (CP) is commonly used in SACs frozen semen. The objective of the study was to evaluate the combined cryoprotective capacity of two permeable CPs at different stages of the cryopreservation protocol in llama semen. Sixteen ejaculates from 4 llama males were analysed, and sperm quality was assayed in raw semen, at 5°C, after equilibration of samples with the CPs and when samples were thawed. The following CPs and combination were used: 6% glycerol (GL), 6% dimethylformamide (DMF) and the combination of both CPs: 3% GL and 3% DMF. A Kruskal-Wallis test and an experimental factorial design, considering one factor with four levels (raw semen, 6% GL, 6% DMF and GL/DMF), were used. Total sperm motility and live sperm with intact acrosomes remained unchanged after equilibration of samples (p > .05). A significant decrease in the percentage of functional membrane, motile and live sperm with intact acrosomes was observed when samples were thawed (GL, DMF and GL/DMF). Nevertheless, the cryopreservation protocols used preserved sperm DNA quality; thus, sperm chromatin condensation and DNA fragmentation were unaffected (p > .05) when GL, DMF and GL/DMF were used. To conclude, no superiority was found between the use of a single or a combination of permeable cryoprotectants to freeze llama semen.

Air-Drying Llama Sperm Affects DNA Integrity.

The objective of this study was to evaluate the effects of air-drying preservation on llama sperm DNA. Semen collections were carried out using electroejaculation under general anesthesia. A total of 16 ejaculates were processed from 4 males (n = 4, r = 4). Each sample was diluted 4:1 in a collagenase solution in TALP media, then incubated and centrifuged at 800 g for 8 min. The pellet was re-suspended to a concentration of 20 million sperm/ml in TALP. Then the samples were placed onto sterile slides forming lines and were left to dry under laminar flow for 15 min. After this, the slides were placed into Falcon centrifuge tubes and kept at 5°C. Sperm characteristics (motility, membrane function, viability and morphology) were evaluated in raw semen and in the air-dried samples kept at 5°C for 30 min. DNA evaluation (integrity and degree of chromatin condensation) was carried out in raw semen and in the air-dried samples after 30 min, 7, 14, 21, 30, and 60 days after preservation. To compare raw semen to the air-dried samples, a Wilcoxon test was used for all sperm characteristics except for DNA, where a paired Student t-test was applied. A split plot design was used to compare chromatin condensation between the different periods of preservation and a Kruskal Wallis test was used to compare DNA integrity. Motility, membrane function, viability and sperm with intact DNA decreased in the air-dried samples (p < 0.05), while morphology and chromatin condensation were not affected (p > 0.05). No significant differences were observed in the percentage of sperm with condensed chromatin between the different periods of preservation (p > 0.05). On the other hand, a significant decrease in the percentage of sperm with intact DNA was observed as from day 7 of preservation (p < 0.05). In conclusion the air-drying process has a negative effect on llama sperm DNA, hence the media used will need to be improved to protect DNA and be able to implement this technique in this species.

Development of a GnRH-PGF2α Based Synchronization and Superstimulation Protocol for Fixed-Time Mating in Llama Embryo Donors.

The aim of the present study was to evaluate the application of a GnRH-PGF2α based synchronization and superstimulation protocol for fixed-time natural mating in llama embryo donors. All females (n = 8) received 8 µg IM of GnRH analog (GnRHa; buserelin) on day 0, regardless of follicular status. After eight days, another GnRHa dose was administered followed by 250 µg IM PGF2α (cloprostenol). A dose of 1000 IU IM of equine chorionic gonadotrophin (eCG) was applied on day 12 and a new dose of PGF2α was administered on day 13. All embryo donors were mated with a male of proven fertility followed by a GnRHa dose on day 18. 24 h later, mating was repeated with a different male. Transcervical uterine flushing for embryo recovery was carried out on all females on day 26. Recipient females received one dose of GnRHa (day 0) two days after the first mating of embryo donor females. A 75% (6/8) of embryo donors responded to the superstimulation treatment with a range of 2 to 5 corpus luteums (CLs) on embryo recovery day. A total of 24 CLs were registered, with a mean of 4 ± 0.9 CLs per female. Embryo recovery rate was 66.7% (16/24), with a range of 0 to 4 embryos and a mean of 2.7 ± 1.5 embryos per female. Regarding quality of the recovered embryos, 56.2% were grade I, 6.2% were grade II and 37.5% were grade V (untransferable; arrested morulae). Grade I and II embryos (n = 10) were transcervically transferred into recipient females (n = 10) six days after inducing their ovulation. At 24 days after embryo transfer (ET), a 50% pregnancy rate was registered. In conclusion, a group of llama embryo donors can be synchronized and superstimulated using a fixed-time mating protocol based on GnRHa, PGF2α, and eCG without the necessity of using ultrasonography in the field.

Incubation of frozen-thawed llama sperm with seminal plasma.

Seminal plasma is intimately connected to sperm physiology and particularly in South American Camelids, has demonstrated to be involved in multiple physiological reproductive events. Different percentages of seminal plasma (0%, 10% and 50%) were added to thawed llama semen samples with the objective of evaluating the interaction with cryopreserved sperm over time (0, 1.5 and 3 hr at 37°C). A total of 20 ejaculates from five adult llama males (n = 5; r = 4) were evaluated. A significant decrease in sperm motility, membrane function and live sperm was observed in all thawed samples (0%, 10% and 50%) at 0 hr when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > .05), but a significant increase in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples versus raw semen. When evaluating thawed samples over time, a significant decrease of motility and membrane function was observed, while the percentages of total live sperm were preserved over the 3 hr of incubation in all final concentrations evaluated. To conclude, the addition of 10% or 50% of seminal plasma was incapable of preserving motility or membrane function of frozen-thawed llama sperm during 3 hr of incubation.