Protocol for obtaining leukocyte- and thrombocyte-rich fibrin (L-TRF) membrane in domestic quail (Coturnix coturnix japonica) Bruna Zafalon‑Silva, Carine Dahl Corcini, Mariana Ferreira Fehy, Antonio Sergio Varela Junior, Raqueli Teresinha França Comparative Clinical Pathology, 2026 Autologous hemoconcentrates, such as leukocyte- and platelet-rich fibrin, have been widely used in regenerative medicine; however, standardized protocols for avian species of experimental and clinical relevance remain scarce. This study aimed to standardize the preparation of leukocyte- and thrombocyte-rich fibrin (L-TRF) membranes in domestic quail (Coturnix coturnix japonica) and to evaluate their clinical and laboratory characteristics. Fourteen adult quail underwent right jugular vein venipuncture, with whole blood collected into additive-free glass tubes and centrifuged at 400 g at approximately 24 °C for 5, 10, 15, and 20 min. Membranes were obtained after partial serum absorption and characterized by light microscopy and transmission electron microscopy. The mean blood volume collected was 0.89 ± 0.37 mL (~ 0.7% of body weight). Consistent viscoelastic membranes were formed between 10 and 20 min of centrifugation. The clots exhibited a layered organization composed of fibrin, a leukocyte–thrombocyte layer, and an erythrocyte-rich fraction. Microscopic analysis demonstrated a three-dimensional fibrin network containing embedded thrombocytes and leukocytes, predominantly heterophils and lymphocytes. In conclusion, centrifugation of domestic quail whole blood at 400 g for 20 min in plain glass tubes, followed by standardized serum absorption, consistently yields cohesive and manageable L-TRF membranes. This reproducible, low-cost protocol contributes to the standardization of hemoconcentrate preparation in avian species and supports future applications in avian regenerative medicine.
First Report of CRISPR-Cas9 Ribonucleoprotein Delivery Into Teleost Spermatozoa With Preserved Membrane and Genomic Integrity Laís dos Santos Gonçalves, William Borges Domingues, Leandro Silva Nunes, Eduardo Bierhals Blödorn, Eduardo Nunes Dellagostin, Amanda Weege da Silveira Martins, Izani Bonel Acosta, Carine Dahl Corcini, Antônio Sergio Varela, Charles Nunes Fróes, Vinicius Farias Campos Molecular Reproduction and Development, 2026 Delivery of large molecular complexes into spermatozoa remains a challenge in reproductive biotechnology. Teleost sperm possess highly compacted chromatin and minimal cytoplasmic volume, limiting intracellular access of ribonucleoprotein assemblies. Here, we evaluated whether CRISPR‐Cas9 ribonucleoprotein (RNP) complexes can be introduced into Nile tilapia spermatozoa using capillary electroporation while preserving cellular integrity. RNP uptake was quantified via EGFP fluorescence, and sperm performance was assessed through motility, membrane integrity, mitochondrial activity, and DNA fragmentation analyses. Approximately 10%–13% of spermatozoa exhibited detectable EGFP signal following electroporation. Increased pulse numbers reduced motility and mitochondrial activity, whereas membrane integrity was preserved and no increase in DNA fragmentation was detected. A single pulse (1250 V, 40 ms) provided the best balance between RNP uptake and functional preservation. These findings support capillary electroporation as a minimally disruptive strategy for CRISPR‐RNP delivery in teleost reproductive biology.
Brief Communication: Increased Frequency of Micronucleus in the Bone Marrow of Mice Under Simulated Occupational Exposure to Isoflurane Marina Zanin, Mariana Gobbo Braz, Maria Vitória Destro, Antonio S. Varela Junior, Martielo Ivan Gehrcke, Carine Dahl Corcini Environmental and Molecular Mutagenesis, 2026 Isoflurane is a widely used inhalational anesthetic. Occupational exposure to waste anesthetic gases (WAGs) raises concerns about DNA damage. Bone marrow, highly susceptible due to its proliferative activity, has been scarcely investigated in rodents after exposure to WAGs. Therefore, the present study evaluated the impact of this exposure on micronucleus (MN) frequency and cytotoxicity, under conditions that mimic work environments with WAG to which thousands of professionals worldwide are occupationally exposed. Swiss mice were randomized into control, exposed, and recovery groups. Animals were subjected to daily 5 h exposures to 50 ppm isoflurane for 30 days. Bone marrow samples were analyzed using the MN assay. Isoflurane‐exposed mice showed a significant increase in micronucleated polychromatic erythrocytes compared to controls ( p < 0.0001). The recovery group exhibited a partial reduction after 20 days without exposure, though values remained above the control. Simulated occupational isoflurane exposure induces chromosomal instability in bone marrow cells, even at recommended safety levels.
Effects of ultraviolet B radiation and cold storage on Ram sperm morphology and physiology Daniele S de S. Cestaro, Antonio Sergio Varela Junior, Anthony Valverde, Ana Paula de Souza Votto, Daza de Moraes Vaz Batista Filgueira, Marc Yeste, Carine Dahl Corcini International Journal of Biometeorology, 2026 Ultraviolet B radiation damages DNA increases oxidative stress and impairs fertility and survival in organisms. The spermatozoa are particularly vulnerable, experiencing altered structure, function, and fertilization capacity due to UVB exposure. The objective of the present study was to evaluate the effects of ultraviolet B radiation and cold storage on the morphology and physiology of ram spermatozoa. The samples of fresh semen from seven animals, obtained during six collections, yielded 42 ejaculates. Aliquots were diluted in Tris egg yolk and were subjected to varying doses of UVB radiation, namely no radiation (0 mJ/cm2), 2.199 mJ/cm2, 4.398 mJ/cm2, 6.597 mJ/cm2, 8.796 mJ/cm2, and 10.995 mJ/cm2. All evaluations were carried out in duplicate and immediately after UVB exposure; samples were also stored at 5 °C for analysis at 24 and 48 h. Sperm were evaluated for motility using a computer assisted sperm analysis system (CASA), apart from plasma membrane integrity, mitochondrial function, DNA integrity, and acrosomal fluorescence. While UVB exposure damaged the acrosome, the plasma membrane and DNA remained intact. Storage at 5 °C for 24 h and 48 h did not affect any of the structures studied, and even though certain aspects of sperm kinematics were affected, they retained motility. Thus, exposure to UVB resulted in lower sperm motility and structures without completely damaging the sperm.
Dairy bull sperm subpopulation behaviour in frozen-thawed semen across breed, temperature, and thawing time F Sevilla, I Araya-Zuniga, JM Solis, C Corcini, P Cervantes-Acosta, A Hernandez-Beltran, R Molina-Montero, D Pichardo-Matamoros, A Valverde Veterinarni Medicina, 2026 Optimising dairy cattle reproduction with assisted reproductive technologies, such as artificial insemination, requires standardised semen handling and analysis. This study evaluated the kinematic structure of the spermatozoan subpopulation in frozen-thawed Holstein and Jersey bull semen doses under different thawing protocols. We used frozen semen doses taken from four bulls of each breed. Nine semen doses were collected from each animal, for a total of 72cryopreserved doses. Straw thawing was performed atthree temperatures (35C, 37C, and 40C) and three times (30s, 40s, and 45s). Sperm kinematic patterns were evaluated using aCASA-mot system (ISAS®v1). Sperm kinematic variables for each breed, temperature, and thawing time identified four subpopulations. The analysis revealed aneffect ofbreed and semen thawing protocols (P<0.05) on sperm subpopulation distribution, sperm movement, and swimming patterns. Subpopulation analyses based on semen assessment are needed to further interpret the relevance and effect on fertility.
Defining optimal frame rate for kinematic semen analysis of different dog breeds assessed by computer-assisted sperm analysis Francisco Sevilla, Marcos Campos, Ignacio Araya-Zúñiga, Almudena García-Molina, Carine Dahl Corcini, Alejandro Saborío-Montero, Carles Soler, Arcesio Salamanca-Carreño, Antonio Sergio Varela Junior, Anthony Valverde Systems Biology in Reproductive Medicine, 2026 v1 system. Videos were acquired at 500 frames per second (fps) and computationally down sampled to 25-400 fps. Sperm kinematic variables were modeled using an exponential regression to estimate the frame rate required to reach 95% of the asymptotic value. Curvilinear velocity (VCL) was the most frame-rate sensitive parameter. The optimal frame rate calculated from the kinematic variable VCL varied among breeds, ranging from 433.72 fps for the Miniature Schnauzer to 939.67 fps for the Golden Retriever. Furthermore, significant differences were found in all analyzed sperm kinematic parameters between breeds. Marked breed effects were detected in motility, concentration and kinematic patterns at 500 fps, with Jack Russell, French Bulldog, Giant Schnauzer and Golden Retriever showing the highest total and fast motility, and Dachshund and Miniature Schnauzer the lowest. Overall, dogs require higher frame rates than most domestic species for reliable CASA-based kinematic assessment, and breed effects should be considered in future research when interpreting canine semen data.
Characterization of isolated late preantral and early antral equine ovarian follicles Ana Normélia P Morais, Gustavo D A Gastal, Francisco L N Aguiar, Kendall A Hyde, Melba O Gastal, Laritza F Lima, Lucy V S Ñaupas, Ana Flávia B Silva, Verónica A B Becerra, Luiza Gheno, Bruna R Curcio, Carine D Corcini, Ana Paula R Rodrigues, José R Figueiredo, Antonio S Varela, Eduardo L Gastal Reproduction and Fertility, 2026 Graphical Abstract Abstract Understanding how to effectively isolate a large number of healthy follicles is essential for increasing success rates in ARTs, such as in vitro culture and cryopreservation. Late secondary (preantral) and early tertiary (antral) follicles isolated from the intermediary portion of equine ovarian tissue sliced at two different thicknesses (0.25 vs 0.50 mm) were characterized according to regional distribution (dorsal, medial, and ventral), morphofunctional features, and epigenetic profiles. After isolation via microdissection, follicles were evaluated for membrane integrity (trypan blue), viability (propidium iodide and Hoechst 33342), mitochondrial membrane potential (JC-1), reactive oxygen species (ROS; H2DCFDA), and histone trimethylation patterns (H3K4me3 and H3K9me3). Regardless of the ovarian tissue slice thickness or region, all isolated follicles had intact membranes, confirming the effectiveness of mechanical microdissection. The key findings from this study reveal that i) recovery of preantral and antral follicles within different ovarian regions is influenced by tissue slicing thickness; ii) the abundance of recoverable follicles varies among the ovarian regions; iii) preantral and antral follicle viability and ROS levels remain consistent between developmental stages, irrespective of tissue slicing thickness, although mitochondrial membrane potential is influenced by slicing thickness; iv) in antral follicles, viability and ROS levels vary depending on location within the ovarian regions; and v) epigenetic markers (H3K4me3 and H3K9me3) exhibit different patterns, depending upon follicular developmental stage. Altogether, these results demonstrate that optimal follicle isolation depends on tissue thickness and ovarian region and that viable, good-quality preantral and antral follicles can be successfully isolated from equine ovarian tissue. Lay summary Follicles are the functional units of the ovary that contain and support the egg. Depending on the presence of a fluid-filled cavity called an antrum, follicles are developmentally classified as preantral (no antrum) or antral (antrum present). In this study, small preantral and antral horse follicles were removed with needles from ovarian tissue sliced at different thicknesses and classified by their location and other characteristics. The health of the harvested follicles was then examined in several ways (cell survival, toxic by-product levels, and certain types of gene control) to evaluate how slice thickness during follicle removal affects these structures. This study reports, for the first time, that tissue slice thickness influences the removal of late preantral and early antral follicles in horses and that the location of these follicles depends on their developmental stage and quality, with these follicles showing distinct levels of gene control.
microRNAs for qPCR Normalization Under Morphofunctional Conditions in Bovine Sperm (Bos taurus) Lucas Petitemberte de Souza, Leandro Silva Nunes, Luana Carla Salvi, Laís dos Santos Gonçalves, Luana Ferreira Viana dos Reis, Izani Bonel Acosta, Carine Dahl Corcini, Antonio Sergio Varela Junior, Fábio Gularte Barreto, Marcelo Brandi Vieira, Diego Corrêa Silveira, Jeaniffer Melgarejo Vieira, Gustavo Freitas Ilha, William Borges Domingues, Vinicius Farias Campos Molecular Reproduction and Development, 2025 Cattle represents one of the most common and widely distributed categories of large ruminants, with well‐established production practices. Fertility is a key factor that significantly influences the success of this production. Studies have shown that microRNAs (miRNAs) present in sperm cells play a crucial role as regulators of processes related to sperm functionality. miRNAs quantification by qPCR is one of the most accurate and straightforward methods, but this technique requires data normalization, and there is no universal consensus on which miRNAs should be used. The present study aimed to identify suitable miRNAs normalizers for qPCR analysis of Bos taurus semen. To achieve this, normalization candidates were assessed under different semen quality conditions, considering sperm morphology and motility. A small nuclear RNA (U6) and six miRNA candidates (Let‐7c‐5p, miR‐100‐5p, miR‐25‐3p, miR‐26a‐5p, miR‐204‐5p, miR‐92a‐3p) were selected. The expression stability of each candidate was analyzed using four independent methods (delta Ct, geNorm, NormFinder, and BestKeeper), under the semen quality conditions. Additionally, a comprehensive stability analysis was conducted using RefFinder, for each condition individually and for the combined conditions. The results indicated that miR‐92a‐3p was the most stable reference miRNA for motility‐related analyses, while Let‐7c‐5p emerged as the best candidate for morphology‐focused analyses. As a normalizer to analyze samples concomitantly, Let‐7c‐5p was identified as the optimal normalizer, while miR‐26a‐5p was the least stable candidate. This study provides the first identification of miRNA normalizers for qPCR analysis of Bos taurus semen, enabling more accurate miRNA quantification in this biological matrix and species.
Environmental contamination and male reproductive health: (ir) reversible effects in child-and adulthood CARINE DAHL CORCINI, ANTONIO SERGIO VARELA JUNIOR, MARC YESTE Anais Da Academia Brasileira De Ciencias, 2025 Infertility affects 10-15% of reproductive-age couples, with causes ranging from genetic factors to unidentified reasons. Environmental conditions, particularly pollutants, play a significant role in male fertility. Yet, public health policies often overlook reproductive health, despite mounting evidence of pollutants’ detrimental repercussion. Understanding this impact is crucial to prevent the effects of dangerous exposure, especially given the high levels of environmental pollutants in today’s world. Most of the previous research about the adverse effects from contaminants has been conducted in rodents, with limited human epidemiological research. This article reviews the evidence about the impact of various contaminants (air pollutants, water contaminants, pesticides, herbicides, radiation, heavy metals, and plastics) on male reproductive health, particularly sperm quality and fertility. The literature suggests that exposure to contaminants during fetal development and childhood has irreversible effects, while those of adult exposure are often reversible. These findings highlight the need to alert society about reproductive health threats from certain contaminants. Public authorities should consider this situation when designing health plans, and individuals envisaging fatherhood should be aware of these risks.
An assessment of Bacillus thuringiensis var. israelensis effects on hematology, intestinal morphology, and testicular growth in chickens Paola de Freitas Feltrin, Aline Arassiana Piccini Roll, Camila Von Mühlen, Norton Luis Souza Gatti, Fábio Pereira Leivas Leite, Carine Dahl Corcini, Antonio Sérgio Varela Júnior, Ana Paula Nunes, Eduardo Gonçalves Xavier, Victor Fernando Büttow Roll Revista Brasileira De Saude E Producao Animal, 2025 This study aimed to evaluate the effects of dietary supplementation with Bacillus thuringiensis var. israelensis (Bti) on hematological parameters, intestinal morphometry, and testicular growth in chickens. A total of 288 Cobb chicks, comprising both males and females, were included in the study and fed diets supplemented with Bti at a concentration of 1x108 CFU per gram of feed for 42 days. The experimental design consisted of four treatments, each with 12 replicates of six birds, as follows: T1: males without Bti; T2: females without Bti; T3: males with Bti; and T4: females with Bti. Bti supplementation significantly reduced crypt depth and increased the villus-to-crypt ratio (P<0.05). However, other variables, including hematological parameters and testicular growth, were not significantly affected. In conclusion, while Bti supplementation improved certain aspects of intestinal morphology, its effects on hematological parameters and testicular growth were minimal in this study.
Occupational Exposure to 50 ppm Isoflurane Impairs Sperm Parameters in Mice Marina Zanin, Antonio Sergio Varela Junior, Izani Bonel Acosta, Edenara Anastacio da Silva, Maria Eduarda Bicca Dode, Martielo Ivan Gehrcke, Mariana Gobbo Braz, Carine Dahl Corcini Journal of Occupational and Environmental Medicine, 2024
Trachemys dorbigni as a metal(loid) bioindicator: a study in rural and urban areas Amanda Andersson Pereira Stark, Carine Dahl Corcini, Marc Yeste Oliveras, Adalto Bianchini, Izani Bonel Acosta, Patrícia Gomes Costa, Gabriel da Silva Zani, Antonio Sérgio Varela Junior, Raqueli Teresinha França Environmental Monitoring and Assessment, 2024
Birthweight leads to seminal and testicular morphofunctional commitment in sexually mature boars Rafaela Costa Rubim Sacramento, Laura Paolucci Neuenschwander Maciel, John Lennon de Paiva Coimbra, Carine Dahl Corcini, Antônio Sergio Varela Junior, Eliza Rossi Komninou, Charis O. Hogg, Cheryl J. Ashworth, Monique de Albuquerque Lagares, Hélio Chiarini-Garcia, Fernanda Radicchi Campos Lobato de Almeida Theriogenology, 2022
Nanocarried antioxidants in freezing extenders for boar spermatozoa S. L. Soares, Camila R. C. Brito, Andreia Nobre Anciuti, Norton C. Gatti, Carine Dahl Corcini, Antonio Sergio Varela, Mariana G. Marques, Francisco N. Fonseca, Eliza R. Komninou, Thomaz Lucia Andrologia, 2021
Oral cytological changes in young adults related to alcohol consumption Merlyn dos Santos Maidana, Antônio Sergio Varela Junior, Carine Dahl Corcini, Jessica Ribeiro Pereira, Diego Martins Pires, Ronan Adler Tavella, Caroline Lopes Feijo Fernandes, Marina dos Santos, Edariane Menestrino Garcia, Flavio Manoel Rodrigues da Silva Júnior Archives of Oral Biology, 2021
High-speed centrifugation of extender of freeze-thaw boar semen Camila R. C. Brito, Antonio Sergio Varela Junior, Stela Mari M. Gheller, Izani B. Acosta, Andreia N. Anciuti, Norton Luiz S. Gatti, Edenara A. Silva, Nathalia W. Knabah, Carine D. Corcini Reproduction in Domestic Animals, 2021
Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome William B. Domingues, Eduardo B. Blodorn, Amanda S.W. Martins, Eduardo N. Dellagostin, Eliza R. Komninou, Joaquin I. Hurtado, Carine D. Corcini, Antonio S. Varela Junior, Luciano S. Pinto, Frederico S. Kremer, Tiago Collares, Danillo Pinhal, Gonzalo Greif, Carlos Robello, Augusto Schneider, Su Guo, Vinicius F. Campos Theriogenology, 2020
Meroanencephaly in an english pointer neonate Luísa Mariano Cerqueira da Silva, Helena Piúma Gonçalves, Thaís Cozza dos Santos, Clarissa Caetano de Castro, Cristina Gevehr Fernandes, et al. Acta Scientiae Veterinariae, 2020
Exogenous ATP in the Cryopreservation of Brycon orbignyanus Spermatozoa Cryo Letters, 2020
Sperm parameters reflect the effects of topical use of Copaifera oil (Copaifera sp.) in Wistar rats Revista Brasileira De Plantas Medicinais, 2020
Male adult rat sperm parameters after skeletal muscle injury Maicon Borges Moraes, Cassio Noronha Martins, Melina Hauck, Douglas Dalcin Rossato, Carine Dhal Corcini, Antonio Sergio Varela Junior, Luis Ulisses Signori Revista Brasileira De Medicina do Esporte, 2019
Roundup® Herbicide Decreases Quality Parameters of Spermatozoa of Silversides Odontesthes Humensis Tony Silveira, Antonio Sergio Varela Junior, Carine Dahl Corcini, William B. Domingues, Mariana Remião, Lucas Santos, Bruna Barreto, Ingrid Lessa, Diego Martins, Robert T. Boyle, Patrícia Gomes Costa, Adalto Bianchini, Ricardo B. Robaldo, Vinicius Farias Campos Bulletin of Environmental Contamination and Toxicology, 2019
Effects of dietary microencapsulated Cymbopogon flexuosus essential oil on reproductive-related parameters in male Rhamdia quelen Carine de Freitas Souza, Cristine Rampelotto, Bruno Bianchi Loureiro, Fernanda Alves Pereira, Adriane Erbice Bianchini, Carine Dahl Corcini, Antonio Sergio Varela Junior, Tatiana Emanuelli, Leila Picolli da Silva, Sílvio Teixeira da Costa, Kalyne Bertolin, Monique Tomazele Rovani, Paulo Bayard Dias Gonçalves, Berta Maria Heinzmann, Bernardo Baldisserotto Fish Physiology and Biochemistry, 2018
Gelatin protects ram semen stored for 72 h at 5 °C Stela Mari Meneghello Gheller, Carine Dahl Corcini, Jorgea Pradieé, Guilherme Rizzoto, Thomaz Lucia, Fabiana Moreira, Antonio Sergio Varela Junior Small Ruminant Research, 2018
Study of fetal equine ovary: An histological approach Gabriela D. de Moraes, Universidade Federal do Rio Grande, Brazil, Bruna da R. Curcio, Carlos E. Nogueira, Fernanda M. Pazinato, Ilusca S. Finger, Alessandra C. Silva, Antônio S. Varela Junior, Carine Dahl Corcini, Universidade Federal de Pelotas, Brazil, UFPel, Brazil, FURG, Brazil, UFPel, Brazil Pesquisa Veterinaria Brasileira, 2016
Vitrification of equine oocytes with a polyvinyl alcohol after in vitro maturation with equine growth hormone and insulin-like growth factor-i Cryo Letters, 2014
