Assisted Reproductive Technologies (ART) are an important tool for accelerating genetic progress in livestock. Because of their particularities, the successful application of ART in camelids is a challenge [1,2]. Artificial Insemination (AI) requires complex interventions such as ultrasound monitoring and artificial induction of ovulation in females, and correct semen handling and cryopreservation of sperm [3]. While some progress has been reported in the hormonal control of female camelids, semen-related technologies are hindered by the lack of reliable methods for semen handling and cryopreservation [4]. Camelid semen is characterized by a highly viscous and low-volume ejaculate, with low sperm concentration and reduced progressive motility. These semen and sperm characteristics make difficult the development of AI protocols. Numerous novel attempts to improve semen handling and sperm cryopreservation have been reported with variable results [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]]. As a consequence, there are few reports of pregnancies in camelids achieved with cooled or frozen-thawed sperm [[19], [20], [21], [22]], which results in low pregnancy rates for widespread use in breeding.

At present, there is consensus that the solution must be approached from a deep knowledge of the species [1,2,23]. The composition of camelids’ seminal plasma is different from other livestock [24,25], and it is possible that different interactions occur between the diluents and the sperm or seminal plasma [3] when sperm are preserved at low temperatures during the cooling or freezing processes [16,26,27]. Information regarding the effect at the molecular level of cryopreservation on sperm of these species is still scarce [28,29]. In previous studies, we reported that cooling and freezing induce ultrastructural alterations to the llama sperm [30] and generate protein modifications such as β-NGF, affecting sperm functionality [29]. To achieve long-term storage of semen for a successful AI, increased knowledge of the molecular aspects of seminal plasma, sperm function, and semen-diluent interactions is required.

In llama sperm, we described a 15 kDa N-acetylgalactosamine-binding protein (SL15). This is a seminal plasma-derived lectin that belongs to the Jacalin-related lectins, a large family of plant and animal lectins. In the llama male, SL15 mRNA expression was found in the testis, bulbourethral glands, and prostate [31]. Although it has not yet been demonstrated, studies suggest that SL15 could have a function during the establishment of sperm reservoirs in the llama oviduct [32,33], preserving sperm viability and fertilizing capacity, as documented for other similar proteins found in bull seminal plasma and boar sperm [34,35]. However, no information about the localization on llama sperm or confirmation of secretion sites for SL15 has been previously reported [31].

In this study, we propose to characterize the presence of SL15 in the llama male reproductive system and sperm; and to determine the effect of cryopreservation on SL15 levels and distribution in llama sperm. We aim 1) to identify the presence and localization of SL15 in the testis, epididymis, prostate, bulbourethral glands, and sperm; and 2) to localize and quantify by flow cytometry SL15 on fresh, cooled, and frozen-thawed llama sperm.