Martha Valdivia

Identifying oocytes with high meiotic competence remains a major challenge for improving in vitro embryo production in South American camelids. Although Brilliant Cresyl Blue (BCB) staining has been used to distinguish alpaca oocytes with different meiotic and developmental competence, the biological and molecular feature associated with their meiotic competence remain poorly understood. This study characterized the transcriptomic profiles of immature alpaca (Vicugna pacos) oocytes classified as BCB-positive (BCB+) or BCB-negative (BCB−) using single-cell RNA sequencing. BCB+ oocytes exhibited a significantly larger ooplasm diameter, a higher proportion of condensed chromatin configurations, and higher in vitro maturation rates than BCB− oocytes. Transcriptomic analysis identified 2978 differentially expressed genes and revealed a clear separation between groups (PC1 = 64.8%). Differentially expressed genes with higher transcript abundance in BCB+ oocytes were enriched in DNA metabolism, cell cycle, oocyte meiosis, and proteasome pathways, whereas those with higher transcript abundance in BCB− oocytes were enriched in gene expression regulation, RNA metabolism, chromatin remodeling, and intracellular signaling. Protein–protein interaction (PPI) network analysis identified distinct hub genes in each group, including CDK1, CCNB2, MAD2L1, and AURKB in BCB+ oocytes and EP300, CREBBP, SMARCA4, and ARID1A in BCB− oocytes. These results provide the first single-cell transcriptomic characterization of BCB-classified alpaca oocytes and establish the molecular basis underlying the biological differences identified by BCB staining.

Cryopreservation of alpaca (Vicugna pacos) epididymal sperm is an important strategy for preserving valuable genetic resources; however, the freeze–thaw process induces oxidative stress and cellular damage that compromise post-thaw sperm quality. This study aimed to evaluate the effects of resveratrol supplementation during cryopreservation on the post-thaw quality of alpaca epididymal sperm. Two hundred ten cauda epididymides were collected, and only sperm samples with progressive motility ≥50% were considered. Sixty epididymal sperm samples met this criterion and were allocated to cryopreservation experiments. Ten samples were used in a preliminary experiment to select the resveratrol concentration for the main experiment, whereas the remaining 50 samples were used in the main cryopreservation experiment. Spermatozoa were evaluated before cryopreservation and after thawing in a TES–Tris egg yolk extender with or without 50 μM Resveratrol. Post-thaw sperm quality was improved with Resveratrol supplementation (P < 0.05), as evidenced by preserved mitochondrial physiology and sperm membrane integrity, reduced oxidative stress, early apoptosis, and DNA fragmentation, and enhanced homologous sperm binding to the zona pellucida. However, oxidoreductase activity did not differ between the cryopreserved groups. In conclusion, supplementing the freezing extender with 50 μM resveratrol improved the post-thaw quality of alpaca epididymal sperm by preserving sperm physiological integrity and reducing oxidative stress and cryopreservation-induced cellular damage.

Among the in vitro embryo production (IVP) procedures, the in vitro maturation (IVM) is an essential step for the oocyte to achieve nuclear and cytoplasmic maturity (competence). In this line, supplementation of the IVM medium with resveratrol have been associated with higher oocyte competence in other species. On the other hand, a precise analysis of transcript levels in mammalian gametes and embryos requires the use of reliable reference genes (RGs) for data normalization. However, RGs specific to IVM of alpaca oocytes have yet to be identified, creating a barrier to functional gene studies in this species. Therefore, this study aimed to identify suitable reference genes to proper analyze gene expression patterns during IVM of alpaca oocytes. Four candidate transcripts (Gapdh, Actb, Rplp0 and Ppia) were selected and their stability during IVM with and without exposure to resveratrol was analyzed using RefFinder software (https://github.com/fulxie/RefFinder). The transcript levels of two target genes (Bmp15 and Sirt1) were normalized by the selected reference genes. Our findings indicate that, among the four candidate reference genes, Rplp0 and Gapdh were the most stable transcripts. Additionally, we observed that transcript levels of Bmp15 were differentially expressed after IVM in the presence of resveratrol (p<0,05). We also demonstrated that supplementation with resveratrol modulates the expression patterns of transcripts associated with chromatin remodeling and folliculogenesis. Thus, this is the first report evaluating suitable reference genes for alpaca oocytes, providing valuable tools to better understand oocyte physiology at the gene expression level of South American camelids.

Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility.

The objective of this study was to evaluate the effect of the addition of atomized black Maca in the cryopreservation medium of epididymal spermatozoa of alpaca species. Fifteen pares of testes and epididymis from alpacas of reproductive age were collected from a local (Huancavelica) slaughterhouse and transported in NaCl 0,9% at 10°C to the laboratory. Each sample of epididymal sperm was divided into six groups: 1) Fresh, 2) Yolk medium (YM), 3) YM+10mg/ml Maca, 4) YM+20mg/ml Maca, 5) YM+30mg/ml Maca, and 6) YM+ Resveratrol (antioxidant positive control). Sperm cryopreservation was performed through the slow-freezing method. Markers associated with functionality such as motility, vitality and plasma membrane integrity. Cellular markers associated with oxidative damage, such as DNA fragmentation, total ROS production and lipid peroxidation, were analyzed before and after freezing. The results showed that the supplementation with black Maca at 20mg/ml improved the sperm motility, viability and integrity of the membrane. Similarly, total ROS production decreased with Maca at 10 mg/ml (25 ± 2.23%) and 20mg/ml (20 ± 2.52%) (P<0.05). However, DNA fragmentation and lipid peroxidation were similar in all groups. These results suggest that Maca at 20mg/ml has a protective effect during the freezing/thawing process of epididymal sperm of alpaca species.

This is the first time that testicular tissue (n = 44) and isolated testicular cells (n = 51) were cryopreserved from alpaca testes 24 h postmortem. For this purpose, internally designed freezing media and cryopreservation protocols were used. Testicular tissue fragments (25 mg) and isolated testicular cells were frozen in MTDB (trehalose and black maca), MTD (trehalose), MSDB (sucrose and black maca), and MSD (sucrose) media. Isolated spermatogonial cells were cryopreserved in two ways, before and after proliferation in vitro. After cryopreservation, the percentage of cell viability in Group 1 (>50% of cell viability) by trypan blue did not show differences within each group (p > 0.05) but showed significant differences when comparing fragments with isolated cells (p < 0.05). Spermatogonial stem cells (SSC) were identified by flow cytometry as strong Dolichos biflorus agglutinin (sDBA) and mitochondrial activity of SSC as strongly positive for MitoSense (sMitoSense+) in intact mitochondria cells, weakly positive for MitoSense (wMitoSense+) in early apoptosis, and necrosis with 7-Aminoactinomycin-D positive (7-AAD). After freezing, in Group 1M (≥30% sMitoSense+), the fragments did not show differences between the media (p > 0.05), but in the isolated cells frozen in MSDB medium, 63.68 ± 8.90% (p < 0.05). In Group 2M (p < 0.05); in sMitoSense+, the isolated cells (34.40 ± 23%) had a higher percentage than the fragments (12.4 ± 5.2) (p < 0.05). On the other hand, MSDB and MSD media were significantly higher for isolated cells than for fragments in sDBA+ (p < 0.05). On the other hand, the SSC (sDBA+) had significant differences (p < 0.05) between fresh cells 7.43 ± 1.3% (sDBA+) compared with those cryopreserved in MSDB medium 1.46 ± 0.34% (sDBA+). Additionally, the proliferated and cryopreserved SSC 6.29 ± 1.17% (sDBA+) did not show significant differences concerning the fresh cells (p > 0.05). In conclusion, the black maca showed antioxidant properties when it was included in the freezing medium and, therefore, improved the SSC's conservation of the alpaca. Furthermore, the proliferation of isolated cells in vitro produces a higher amount of SSC after thawing them for further preclinical or clinical work.

The efficiency of producing embryos using in vitro technologies in livestock species rarely exceeds the 30–40% threshold, indicating that the proportion of oocytes that fail to develop after in vitro fertilization and culture is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting blastocyst yield, the precise identification of noninvasive cellular or molecular markers that predict oocyte competence is of major interest to research and practical applications. The aim of this review was to explore the current literature on different noninvasive markers associated with oocyte quality in the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, large number of surrounding cumulus cells, slightly expanded investment without dark areas, large oocyte diameter (>120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated with better competence. In addition, the combination of oocyte and zygote selection via brilliant cresyl blue (BCB) test, spindle imaging, and the anti-Stokes Raman scattering microscopy together with studies decoding molecular cues in oocyte maturation have the potential to further optimize the identification of oocytes with better developmental competence for in-vitro-derived technologies in livestock species.

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