María Ignacia Carretero

South American camelids (SAC), including llamas ( Lama glama ) and alpacas ( Vicugna pacos ), exhibit distinctive reproductive features, such as induced ovulation and highly viscous seminal plasma (SP), that complicate conventional semen collection and objective sperm evaluation. Consequently, spermatozoa recovered from the cauda epididymis or through surgical vas deferens deviation (VDD) have become valuable alternatives for research and germplasm preservation. These sperm sources also provide a unique opportunity to investigate sperm physiology in the absence of SP. This review critically compares the functional characteristics of ejaculated, epididymal and VDD‐derived spermatozoa and examines how methodological variables, including collection procedures, post‐mortem interval, tissue handling and surgical protocols, contribute to variability among studies. Current evidence indicates that many functional differences observed among sperm sources, particularly in motility and plasma membrane functionality, are largely associated with the presence or absence of SP rather than intrinsic differences in sperm quality. These findings support a reinterpretation of SP as a physiological regulator of sperm quiescence, membrane stability and functional competence rather than merely a technical obstacle. We also review advanced analytical approaches, including flow cytometry and imaging flow cytometry, highlighting their potential to improve the evaluation of camelid sperm physiology and cryobiology, while summarizing current evidence on the developmental competence of sperm from different anatomical sources for in vitro embryo production. Overall, this review provides an updated synthesis of the literature, identifies major methodological limitations and highlights priorities for future research to advance reproductive biotechnologies in SAC.

Background: There are no reports evaluating the effect of antioxidant supplementation during semen cooling in llamas. This study evaluates the impact of zinc oxide nanoparticles (ZnO-NPs) on sperm ultrastructure and characteristics in cooled llama semen. Methods: Eighteen ejaculates obtained from six male llamas were divided into three aliquots and diluted with: AndroMed®-20% egg-yolk (AM-EY); AM-EY with 50 μg/ml of ZnO-NPs; and AM-EY with 100 μg/ml of ZnO-NPs. Samples were cooled to 5°C in an Equitainer®. Evaluations were carried out in raw semen, after dilution (pre-cooling; 0 h), and after 24 and 48 h of storage. Additionally, cooled samples were evaluated after 15, 90, and 180 min of incubation at 37°C. Data were analyzed using Friedman tests, conventional ANOVA, or split-plot design models, depending on the structure and distribution of the data. Results: Oscillatory sperm motility remained unchanged after 24 or 48 h of cooling (P > 0.05). Progressive sperm motility (PM) was similar between all samples cooled for 24 h and pre-cooling samples. A significant decrease in PM was observed in all cooled samples for 48 h compared to pre-cooling samples with ZnO-NPs. Total sperm motility also decreased in cooled samples, particularly after 48 h of storage. No significant differences were found in sperm membrane functionality, morphology, or DNA integrity between raw semen and samples cooled for 24 and 48 h. Regarding viability and acrosomal integrity and intact DNA, no changes were observed among the cooled samples at any of the evaluated storage times (24 and 48 h), nor compared to pre-cooling samples. Live lipoperoxidation-negative sperm decreased in all cooled semen samples compared to raw semen, showing no differences among cooled samples. Plasma membrane damage was evident in all cooled samples, more clearly seen with transmission electron microscopy. Finally, ZnO-NPs did not improve the preservation of cooled samples at 37°C for 180 min. Conclusions: The addition of ZnO-NPs at 50 or 100 μg/ml to AM-EY extenders does not fully protect llama sperm from cooling damage. Further evaluation using other concentrations may be necessary to determine the optimal dose of ZnO-NPs for cooling llama semen. Exploring other nanoparticles or antioxidants could be beneficial. Electron microscopy shows notable structural damage in all samples (with and without ZnO-NPs), highlighting the sensitivity of llama spermatozoa to cooling.

The aim of this study was to evaluate alpaca pregnancy outcomes and birth rates of females inseminated with frozen semen using two commercial extenders. A total of 18 ejaculates from 8 adult alpaca males were obtained with artificial vagina, and macroscopic and microscopic semen characteristics were assessed. Afterwards, samples were divided into two aliquots, diluted with Biladyl® B or AndroMed®, and cooled for 2 h at 5°C. At that moment, sperm motility was evaluated, and samples were frozen through a gradual descent of temperature using a liquid nitrogen tank. To analyse frozen sperm quality, samples were thawed at 38°C for 30 s. Even though a significant decrease in sperm motility and viability was detected when thawed (p < .05), no superiority was found between the two commercial extenders (Biladyl® B vs. AndroMed®). A total of 36 alpaca females were artificially inseminated (AI) between 30 and 34 h post‐injection of a GnRH analogue, administered when a growing dominant follicle was detected through transrectal palpation and ultrasonography. Obtained pregnancy rates were similar between Biladyl® B (33.3%, 6/18) and AndroMed® (22.2%, 4/18). No significant differences were detected in birth rates between the two tested extenders, obtaining 4 and 3 births for Biladyl® and AndroMed®, respectively. In conclusion, alpaca pregnancies and alive offspring can be obtained through AI with frozen semen at similar efficiency rates using commercial diluents, Biladyl® B or AndroMed®.