Abstract
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.
Highlights
It is well-known that South American camelid (SAC) semen presents special rheological characteristics such as high structural viscosity [1] and the capacity to form a thread when handled [2]
Propidium iodide (PI), 6-carboxyfluorescein diacetate, dimethyl sulfoxide, and the reagents for the Tyrode’s albumin lactate pyruvate (TALP) medium and for the hypoosmotic swelling (HOS) test were purchased from Sigma Chemicals (Sigma Aldrich, Buenos Aires, Argentina)
Frozen-Thawed Semen (Control) vs. Frozen-Thawed Samples Processed by Androcoll-ETM
Summary
It is well-known that South American camelid (SAC) semen presents special rheological characteristics such as high structural viscosity [1] and the capacity to form a thread when handled [2]. Ejaculated sperm of these species have a particular motility pattern, they possess oscillatory motility with practically no progressive motility [2,3,4]. Due to these characteristics, several in vitro SAC embryo production studies have reported the use of sperm recovered from animals with deviated deferent ducts or collected from the epididymis after death or castration, since those samples are free of seminal plasma (SP) and have sperm with progressive motility [5,6,7,8,9,10,11,12,13]. It is recommended to wash and centrifuge sperm after using this colloid, increasing the time required for preparation of samples [17] and producing cell damage due to the rise in production of reactive oxygen species [18]
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