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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., (© 2020 Blackwell Verlag GmbH.)

In South American camelids, sperm survival is low after thawing and poor results are obtained when artificial insemination is performed with cryopreserved semen. The aim of this study was to evaluate the effect of different percentages (10% and 50%) of seminal plasma added prior to the process of cryopreservation and also to evaluate the absence of seminal plasma on llama sperm survival after freezing and thawing. A total of 15 ejaculates from five adult llama males (n = 5; r = 3) were evaluated. A significant decrease in sperm motility, viability, membrane function and intact acrosomes was observed in thawed samples (0%, 10% and 50%) when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > 0.05), but a significant increase (p < 0.05) in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples compared to raw semen. Higher percentages of total and progressive sperm motility were observed when 0% and 10% of seminal plasma were used compared to 50%. However, no statistical differences were established for sperm viability, membrane function, morphology, acrosome status and DNA quality between thawed treatments. To conclude, neither of the percentages of seminal plasma used showed superiority nor cryoprotective effect on llama sperm survival., (© 2019 Blackwell Verlag GmbH.)

The objectives of this study were to evaluate the effect over time of different percentages of seminal plasma (SP) on llama sperm characteristics in raw semen and correlate the techniques routinely used to evaluate sperm viability and acrosome status with the Fluorescein Isothiocyanate -Arachis hypogea agglutinin/Propidium Iodide (FITC-PNA/PI). Eighteen ejaculates, obtained from 6 male llamas using electroejaculation, were incubated in 0.1% collagenase in HEPES-TALP (HT), centrifuged and resuspended with SP and HT: 0, 10, 50 and 100% SP. Samples were incubated (37 °C) until evaluation at 0; 1.5 and 3 h. Split plot and factorial designs were used to analyze sperm motility, viability, membrane function and acrosome status and Spearman's test was used for correlation. At 0 h, samples with 100% SP showed oscillatory motility; whereas in samples with 0 and 10% SP, progressive motility was predominant. Viability, membrane function and total motility decreased significantly at 3 h of incubation in samples with 100% SP. Sperm with intact acrosomes were fewer in 0% SP media at all times. FITC-PNA/PI correlated with 6-Carboxyfluorescein Diacetate and Propidium Iodide (CFDA/PI) and with Coomassie Blue (CB) stains (r = 0.8; p = 0.0 and r = 0.5; p = 0.0 respectively)., Conclusions: the motility pattern of llama sperm is influenced by the concentration of SP. The use of SP as the only medium is not able to maintain sperm motility, viability and membrane function for 3 h. A certain percentage of SP is necessary in the medium to avoid spontaneous acrosome reactions. The correlations observed could help to shorten evaluation times and reduce costs in sperm laboratories., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.1% collagenase and then were divided into two aliquots. One was extended in lactose and egg yolk (LEY) (Protocol A: collagenase and SP present). The other aliquot was centrifuged, and the pellet was resuspended in LEY (Protocol B: collagenase and SP absent). Both samples were maintained at 5°C during 24 hr. Routine and DNA evaluations were carried out in raw and cooled semen. Both cooling protocols maintained sperm viability, membrane function and DNA fragmentation, with Protocol A showing a significantly lowered total and progressive motility (p < .05) and Protocol B showing a significant increase in chromatin decondensation (p < .05). Protocol A avoids centrifugation, reducing processing times and making application in the field simpler. However, as neither protocol showed a significant superiority over the other, studies should be carried out in vivo to evaluate the effect on pregnancy rates of the presence of collagenase and SP in semen samples prior to either cooling or freeze-thawing., (© 2016 Blackwell Verlag GmbH.)

The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose-egg yolk (L-EY), (2) Tris-citrate-fructose-egg yolk (T-F-EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5 h and remained at that temperature during 24 h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L-EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A-D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa, cooled to 5°C and kept for 24 h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24 h. These groups (A-C) were inseminated between 22 and 24 h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24 h and AI was carried out within 2 h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The interest for South American camelids has increased in the last years. The aim of the present research was to compare the in vitro production of Lama glama embryos using two techniques: in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). For IVF technique, we compared the effect of adding or not, heparin, penicillamine and hypotaurine as sperm capacitating agents. In the oocyte group subjected to ICSI, activation with or without, ionomycin and 6-dimethylaminopurine (6-DMAP) was assessed. Semen samples were obtained by electroejaculation and incubated at 38 degrees C in a 25% (v/v) collagenase solution. The cleavage and embryo development rates were compared between the different experimental groups. Only the number of cleaved oocytes was less when ICSI with no activation was used (p<0.05).