Abstract
This study compared protocols for cryopreservation of ejaculated, papain-treated alpaca spermatozoa. This included different concentrations of egg yolk (EY; 5, 10 or 15%) and glycerol (2, 5 or 10%), diluent types (SHOTOR, lactose, skim milk or INRA-96™), freeze rates (2, 4 or 8 cm above liquid nitrogen; LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels (pellets, 0.25 mL straws or 0.5 mL straws). Spermatozoa were assessed pre-freeze and 0, 30, 60 and 90 min post-thaw. Forty-one hembras were inseminated with either fresh, papain-treated or frozen-thawed spermatozoa. Motility was affected by EY concentration (P < 0.001), diluent type (P < 0.001), freeze rate (P = 0.003) and storage vessel (P = 0.001). Viability was affected by EY concentration (P < 0.001), diluent type (P < 0.001), storage vessel (P = 0.002) and thaw rate (P = 0.03). For artificial insemination (AI), semen was diluted 1:3 in a lactose-based diluent, with 5% EY and glycerol. Freezing was in 0.5 mL straws, 2 cm above LN for 4 min then thawing at 37 °C for 1 min. Pregnancy rates of those ovulated (n = 26) were not different (1/5 fresh, 1/4 papain-treated, 0/17 frozen-thawed; P = 0.10). Pregnancy can be achieved after AI with papain-treated spermatozoa. Further work is needed to determine the optimal dose, timing and location for insemination.
Highlights
LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels
Post-thaw, spermatozoa frozen in medium containing 5% egg yolk had higher motility (34.17 ± 3.81%) than both 10% (27.92 ± 3.03%) and 20% egg yolk (15.83 ± 2.57%; P < 0.001), this trend continued until 60 min post thaw, when 20% egg yolk had significantly lower motility than the other two treatments
Sperm viability was significantly higher at 5% (36.20 ± 3.51%) and 10% egg yolk (37.30 ± 3.42%) compared to 20% egg yolk (31.42 ± 2.82%; Fig. 1B; P < 0.001) and viability declined over time (P < 0.001)
Summary
LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels (pellets, 0.25 mL straws or 0.5 mL straws). Kershaw-Young and Maxwell[2] found that the viscosity in alpaca seminal plasma is caused by the protein mucin 5B and that by adding the protease papain to alpaca semen, viscosity could be reduced to zero, without adversely affecting the motility, acrosome integrity, viability and DNA integrity of the spermatozoa[3]. Et al.[1] diluted epididymal alpaca spermatozoa (to bypass the issue of viscous seminal plasma) with citrate- Tris- and lactose-based diluents and froze in straws and pellets. They found that a lactose-based diluent, combined with freezing in straws rather than pellets was most effective at retaining acrosome integrity and www.nature.com/scientificreports/. In Experiment 1 and 2 of the current study we aimed to test a wider range of glycerol (2, 5 or 10%) and egg yolk (5, 10 or 20%) concentrations than studied previously
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