Cooling of alpaca spermatozoa using an extender with the addition of different percentages of seminal plasma.

The objective of this study was to evaluate the effect of the addition of different percentages of seminal plasma (SP) during the cooling at 5 °C of alpaca spermatozoa from vas deferens. Fifteen pools of sperm from vas deferens were evaluated and then divided into four aliquots that were diluted to a final concentration of 30 × 106 sperm/ml with either: (1) Tris with 20% egg yolk (T-EY) (control, 0% SP), (2) T-EY with 10% SP, (3) T-EY with 25% SP, and (4) T-EY with 50% SP. Samples were cooled at 5 °C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. A significant decrease in progressive and total sperm motility was observed in samples cooled with 50% SP with respect to all diluted samples, while these parameters were preserved in samples cooled with 0%, 10%, and 25% SP. The percentages of sperm viability, normal morphology, and highly condensed chromatin did not change after the cooling process and were similar between cooled samples. Although a significant decrease was observed in the percentage of spermatozoa with functional membranes and with an intact acrosome in all refrigerated samples compared to raw sperm, the greatest decrease was observed in samples cooled with 50% SP. No advantage was observed from the addition of SP to alpaca spermatozoa obtained from vas deferens and being cooled. In addition, to preserve the sperm motility of cooled samples for up to 72 h, it should be recommended to include a 10% SP in the extender.

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.

Comparison of Extenders With the Addition of Egg Yolk for Cooling Alpaca Sperm Obtained From Deferent Ducts.

The use of non-commercial and commercial extenders for cooling alpaca sperm has already been reported, the latter showing certain advantages over the first. The Andromed® (AM) extender was created for use in ruminants and has also been tested in ejaculated and epididymal alpaca sperm. According to the manufacturer, this extender does not need the addition of egg yolk (EY); however, it is known that the addition of EY to some extenders improves the preservation of cooled sperm. The objective of this study therefore was to compare a non-commercial extender (Tris) with the addition of EY vs. the commercial extender AM with and without the addition of EY, for cooling alpaca sperm obtained from diverted deferent ducts. Fifteen pools of deferent duct sperm were formed using samples from two or three different males for each. Each sperm pool was evaluated and then divided into three aliquots that were diluted to a final concentration of 30 × 106 sperm ml-1 (0 h) with either: (1) Tris with 20% EY (T-EY), (2) AM, or (3) AM with 20% EY (AM-EY). Samples were cooled to 5°C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. The samples that best preserved progressive and total sperm motility at the 24 and 48 h evaluation periods were the ones diluted with AM-EY, observing that with this extender these motility patterns decreased significantly after 72 h of storage compared to time 0 h (p < 0.05). A significant decrease (p < 0.05) in total and progressive motility was observed at 48 h for the T-EY and AM extender compared to 0 h. AM was the only extender in which the percentages of viable sperm decreased significantly (p < 0.05) after 48 h of conservation. For the rest of sperm parameters evaluated, no significant differences were observed between any of the extenders at any evaluation time. The Andromed® extender with the addition of 20% EY could be an alternative option for cooling alpaca sperm obtained from deferent ducts.

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.

Use of Androcoll-ETM to Separate Frozen-Thawed Llama Sperm From Seminal Plasma and Diluent.

It is not easy to separate frozen-thawed South American camelid sperm from seminal plasma (SP) and diluents to be used for in vitro embryo production. The objective of this study was to evaluate Androcoll-E™ (AE) efficiency to separate llama sperm from SP and freezing extender in frozen-thawed semen. A total of 22 ejaculates from five Lama glama males were collected using electroejaculation. After performing semen analysis (sperm motility, concentration, viability, membrane function, and acrosome integrity), samples were cryopreserved with a diluent containing lactose, ethylenediaminetetraacetic acid (EDTA), egg yolk, and 7% dimethylformamide. After thawing, samples were divided in aliquots, one of which was used as a control and the others processed by AE. Experiment 1 (12 ejaculates): 100 µl of frozen-thawed semen was placed on top of 1,000 µl AE column and centrifuged at 800 g for 10 min. Experiment 2 (10 ejaculates): two samples of 100 µl of frozen-thawed semen were placed on two columns of 500 µl AE each, and both were centrifuged at 800 g for 10 and 20 min, respectively. Pellets were resuspended in Tyrode's albumin lactate pyruvate (TALP) medium, and sperm parameters were evaluated. A significant decrease in all sperm parameters was observed in thawed samples compared to raw semen. AE allowed the separation of frozen-thawed sperm from SP and freezing extender independently from the height of the column used and time of centrifugation assayed. Although no significant differences were found between AE columns, higher sperm recovery was observed with 500 µl of AE coupled with 20 min of centrifugation. Despite the significant decrease observed in sperm motility in AE samples, no changes in sperm viability, membrane function, and acrosome integrity were observed when comparing control thawed semen with the sperm recovered after AE (p > 0.05). The use of AE columns, either 500 or 1,000 µl, allows the separation of frozen-thawed llama sperm from SP and freezing extender, preserving the viability, membrane function, and acrosome integrity. Of the protocols studied, 800 g centrifugation during 20 min using a 500 µl column of AE would be the method of choice to process frozen-thawed llama semen destined for reproductive biotechnologies.