Gloria Levano

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.

The COVID-19 pandemic circumstances have varied the pathogens related to acute respiratory infections (ARI), and most specialists have ignored them due to SARS-CoV-2’s similar symptomatology. We identify respiratory pathogens with multiplex PCR in samples with presumptive SARS-CoV-2 but negative RT-qPCR results. We performed a retrospective transversal study employing clinical data and nasopharyngeal swab samples from patients with suspected clinical SARS-CoV-2 infection and a negative PCR result in a private laboratory in Lima, Peru. The samples were analyzed using the FilmArray™ respiratory panel. Of 342 samples, we detected at least one pathogen in 50% of the samples. The main ones were rhinovirus (54.38%), influenza A(H3N2) (22.80%), and respiratory syncytial virus (RSV) (14.04%). The clinical characteristics were sore throat (70.18%), cough (58.48%), nasal congestion (56.43%), and fever (40.06%). Only 41.46% and 48.78% of patients with influenza met the definition of influenza-like illness (ILI) by the World Health Organization (WHO) (characterized by cough and fever) and the Centers for Disease Control and Prevention (CDC) (characterized by fever and cough and sore throat), respectively. A higher prevalence of influenza was associated with ILI by WHO (aPR: 2.331) and ILI by CDC (aPR: 1.892), which was not observed with other respiratory viruses. The clinical characteristic associated with the increased prevalence of rhinovirus was nasal congestion (aPR: 1.84). For patients with ARI and negative PCR results, the leading respiratory pathogens detected were rhinovirus, influenza, and RSV. Less than half of patients with influenza presented ILI, although its presence was specific to the disease.

The identification system of spermatogonial stem cell (SSC) was established in alpaca using the molecular expression as well as the reactivity pattern to Dolichos biflorus agglutinin (DBA) by flow cytometry. Twenty‐four testicles with their epididymis were recovered from adult alpacas at the slaughterhouse of Huancavelica—Perú. Samples were transported to the Laboratory of Reproductive Physiology at Universidad Nacional Mayor de San Marcos. Testes were selected for our study when the progressive motility of epididymal spermatozoa (ESPM) was above 30%. Isolation of SSC was performed with two enzymatic digestions. Finally, sperm viability was evaluated by means of the trypan blue vital stain in spermatogonial round cells. Samples with more than 80% viability were selected. Isolated cells cultured for 2 days were used for identifying the presence of SSCs by the expression of integrin β1 (116 bp) and PLZF (206 bp) genes. Spermatogonia were classified according to the DBA reactivity. Spermatogonia with a strong positive to DBA (sDBA⁺) were classified as SSC (Mean ± SEM=4.44 ± 0.68%). Spermatogonia in early differentiation stages stained weakly positive with DBA (wDBA⁺) (Mean ± SEM=37.44 ± 3.07%) and differentiated round cells as DBA negative (Mean ± SEM=54.12 ± 3.18%). With the use of molecular and DBA markers, it is possible to identify easily the spermatogonial stem cells in alpaca.

The aim of this study was to evaluate the effect of Lepidium meyenii (maca) in chemically and physically subfertile mice. After 35 days, the following groups of mice were evaluated: control, sham, chemical subfertility, chemical subfertility–maca-supplemented, physical subfertility, physical subfertility–maca-supplemented and maca-supplemented only. Motility (32.36% ± 5.34%) and sperm count (44.4 ± 5.37 × 10⁶/ml) in the chemically and physically subfertile mice (11.81% ± 4.06%, 17.34 ± 13.07 × 10⁶/ml) decreased compared to the control (75.53% ± 2.97% and 57.4 ± 19.6 10⁶/ml) and sham (53.5% ± 7.86% and 58.4 ± 14.10 10⁶/ml). Maca was able to reverse the deleterious effect of motility (76.36 ± 1.97) as well as sperm count (53.5 ± 9.18 × 10⁶/ml) on chemical subfertility. In contrast, maca did not reverse the effects of induced physical subfertility nor motility (18.78% ± 14.41%) or sperm count (20.17 ± 11.20 × 10⁶/ml). The percentage of sperm DNA fragmentation in the physically subfertile mice increased (11.1% ± 19.29%) compared to the control (0.84% ± 0.85%). However, in the physically subfertile group, maca decreased sperm DNA fragmentation (2.29% ± 2.30%) closer to the sham (1.04% ± 0.62%) and the control (0.84% ± 0.85%). The group supplemented only with maca showed 0.54% ± 0.50% of spermatozoa with DNA fragmentation. Yet, the differences observed were statistically not significant. In conclusion, it appears that maca activates the cytochrome P450 system after chemically induced subfertility. However, it does not reverse the low mitochondrial membrane potential in spermatozoa compromised in the physical subfertility group.