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This review describes the first steps necessary to apply any reproductive biotechnology in South American camelids (SAC) semen or sperm: sample collection, evaluation and handling. In camelids, the length and position adopted for mating and the site of semen deposition have conditioned semen collection methods. The advantages and disadvantages of available collection methods are summarized. The two main drawbacks for applying assisted reproductive techniques in SAC: sperm concentration and rheological characteristics are discussed. Techniques currently available to reliably evaluate diverse sperm characteristics are described, as are different methods to improve semen handling. Finally, advances made regarding the role of seminal plasma in SAC spermatozoon physiology are addressed. Part II of the review will cover the subsequent steps of dilution and cryopreservation of samples. Current results obtained using artificial insemination (AI) in SAC will also be covered in Part II., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 × 10 6 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., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest None of the authors have any conflict of interest to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate effects of dehydration on sperm DNA with the aim of eventually using this method for preserving llama spermatozoa. Two experiments were conducted: 1) sperm preservation at 5 °C for 60 days in different hyperosmotic solutions (500, 800, 1000 and 1200 mOsmol/l) (n = 6, replications = 2) and 2) sperm preservation at 5 and -20 °C for 60 days in the same hyperosmotic solutions, with supplementary antibiotics (n = 6, replications = 2). Sperm motility, membrane functional integrity, viability and morphology were evaluated at 0 and 48 h of the preservation period (Experiment 1) and at 30 min and 24 h (Experiment 2). Sperm DNA was evaluated at 0 or 30 min (Experiment 1 and 2, respectively) and on days 7, 14, 21, 30 and 60 of the preservation periods. Motility, membrane functional integrity and viability were less when sperm were dehydrated, while sperm cell morphology was not affected. There was a smaller percentage of sperm with condensed chromatin as duration of the preservation period increased when stored in the different hyperosmotic solutions. There was a markedly smaller (P < 0.05) percentage of sperm with intact DNA in all solutions as the duration of preservation increased, with there being greater values for intact DNA at -20 °C than sperm preserved at 5 °C. Llama sperm chromatin condensation was slightly affected by the process of dehydration. There was a markedly smaller percentage of sperm with intact DNA in the dehydrated semen samples., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ovulation of South American Camelids is induced by mating. After copulation, sperm are stored into the oviduct to be released near ovulation time. To study whether copulation induces matrix metalloproteinase-2 (MMP2) secretion in the oviduct, the occurrence of MMP2 in oviductal tissue and oviductal fluid (OF) from 24 h post-mated was compared with non-mated llama females. There was an incremental increase of MMP2 in the oviductal epithelial cells, and MMP2 activity in OF after copulation. Additionally, MMP2 activator (MMP14), inducer (EMMPRIN) and inhibitor (TIMP2) were present in the oviductal epithelial cells of both non-mated and post-mated females. A post-mating segment-specific regulation occurred because relative abundance of TIMP2 mRNA was greater in the utero tubal-junction which was accompanied with a reduced amount of MMP14 in the ampulla in comparison with the non-mated females. To examine the effect of MMP2 on semen liquefaction and sperm physiology, the effects of addition of recombinant human MMP2 was evaluated. The MMP2 had no effect on semen thread formation and seminal plasma protein profile. Sperm viability and plasma membrane function were not influenced by the enzyme treatment either. In summary, in llamas the oviductal microenvironment changes in response to stimuli induced by copulation, increasing the production and secretion of MMP2., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.)

The objective of this study was to compare the efficiency of different sperm selection methods applied to the same llama ejaculate. Four treatments were compared: two variants of the swim up technique (with and without seminal plasma), and two different colloids, Androcoll-E-Large and Percoll(®). Using electroejaculation, 21 semen samples were obtained from 7 llama males (n=7, r=3). The ejaculates were incubated in a solution of 0.1% collagenase, to decrease thread formation, and then split into 4 aliquots: one aliquot was layered over a column of Androcoll-E-Large (SLC) and the second over a column of Percoll (45%). The third aliquot was deposited in a tube with culture medium and was incubated at a 45° angle for 30min at 37°C (SU1). The last aliquot was centrifuged to separate the spermatozoa and seminal plasma. The sperm pellet obtained was resuspended, and transferred to a tube with culture medium which was incubated at an angle of 45° for 30min at 37°C (SU2). Both aliquots SLC and P showed higher proportions of progressive motility and plasma membrane functionality (p≤0.05) than raw semen. There were no significant differences (p>0.05) in sperm viability and in normal spermatozoa between raw semen and treatments. Nevertheless, only SLC did not have a significant increase of bent tails. In conclusion SLC centrifugation would be the method of choice for selecting llama spermatozoa., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP-BSA medium. An aliquot was sonicated to remove the acrosomal content (positive control). The rest of the sample was incubated for 3h at 38 °C with 5% CO2 and 100% humidity., Treatments: Three aliquots were further incubated 1h with one of the following AR inducers: calcium ionophore, ionomycin or progesterone., Controls: One without inducers and the other, incubated with dimethyl sulfoxide (vehicle of the inducing agents). Acrosomes were evaluated at time 0 and after 4h incubation. Calcium ionophore was the most potent agent for inducing the AR (67.2 ± 14.4% live+dead AR sperm) (P < 0.05). These samples showed no motility and viability was very low (0-30%). Both ionomycin and progesterone presented significantly higher (P < 0.05) percentages of total AR sperm than the controls, but had similar percentages of dead reacted sperm to the controls. A positive correlation was observed between the intact acrosome FITC-PNA/PI pattern (live+dead sperm) and the acrosome-present CB pattern (r = 0.64; P = 0.000) in all the evaluated samples., Conclusions: the FITC-PNA/PI technique simultaneously evaluates viability and acrosomal status in llama spermatozoa and calcium ionophore could be used as a control of AR., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm., (Copyright © 2012 Elsevier B.V. All rights reserved.)