ABSTRACT Introduction Obstructive azoospermia occurs when there is a blockage in the male reproductive tract, leading to a complete absence of sperm in the ejaculate. It constitutes around 40% of all cases of azoospermia (1, 2). Blockages in the male reproductive tract can arise from either congenital or acquired factors, affecting various segments such as the epididymis, vas deferens, and ejaculatory ducts (3). Examples of congenital causes encompass conditions like congenital bilateral absence of the vas deferens and unexplained epididymal blockages (4). Acquired instances of obstructive azoospermia may result from factors like vasectomy, infections, trauma, or unintentional injuries caused by medical procedures (5). This complex condition affecting male fertility, presents two main treatment options: microsurgical reconstruction and surgical extraction of sperm followed by in vitro fertilization (IVF). Microsurgical reconstruction proves to be the most cost-effective option for treating obstructive azoospermia when compared with assisted reproductive techniques (6, 7). However, success rates of reconstruction defined by patency are as high as 99% for vasovasostomy (VV) but decline to around 65% if vasoepididymostomy (VE) is required (8, 9). Thus, continued refinement in technique is necessary in order to attempt to improve patency for patients undergoing VE. In this video, we show a comprehensive demonstration of microsurgical VE, highlighting the innovative epididymal occlusion stitch. The goal of this innovative surgical technique is to improve outcomes for VE. Materials and Methods The patient is a 39-year-old male diagnosed with obstructive azoospermia who presents for surgical reconstruction via VE. His partner is a 37-years-old female with regular menstrual cycles. The comprehensive clinical data encompasses a range of factors, including FSH levels, results from semen analysis, and outcomes from testicular sperm aspiration. This thorough exploration aims to provide a thorough understanding of our innovative surgical technique and its application in addressing complex cases of obstructive azoospermia. Results The procedure was started on the right, the vas deferens was identified and transected. The abdominal side of the vas was intubated and a vasogram performed, there was no obstruction. There was no fluid visible from the testicular side of the vas for analysis, thus we proceeded with VE. Upon inspection of the epididymis dilated tubules were identified. After selecting a tubule for VE, two 10-0 nylon sutures were placed, and it was incised. Upon inspection of the fluid motile sperm was identified. After VE, we performed a novel epididymal occlusion stitch technique. This was completed distal to the anastomosis by placing a 7-0 prolene through the tunica of the epididymis from the medial to lateral side. This stitch was then tightened down with the goal to largely occlude the epididymis so that sperm will preferentially travel through the anastomosis. The steps were then repeated on the left. At 3-month follow up, the patient had no change in testicular size as compared with preoperative size (18cc), he had no testicular or incisional discomfort, and on semen analysis he had presence of motile sperm. After 3 months post-surgery, the patient had motile sperm seen on semen analysis. Discussion The introduction of a novel epididymal occlusion stitch demonstrates a targeted strategy to enhance the success of microscopic VE. Encouragingly, a 3-month post-surgery follow-up reveals the presence of motile sperm, reinforcing the potential efficacy of our approach. This is promising given the historical lower patency, delayed time to patency, and higher delayed failure rates that patients who require VE experience (10). In total, 40% of all azoospermia cases can be attributed to obstruction. The conventional treatments for obstructive azoospermia involve microsurgical reconstruction and surgical sperm retrieval followed by IVF. While microsurgical reconstruction has proven to be economically viable, the quest for enhanced success rates has led to the exploration of innovative techniques. Historically, the evolution of VV and VE procedures, initially performed in the early 20th century, laid the foundation for contemporary microsurgical approaches (11). Notably, the microscopic VV demonstrated significant improvements in patency rates and natural pregnancy likelihood, as evidenced by the seminal Vasovastomy Study Group study in 1991 (8). In contemporary literature, success rates particularly for VE remain unchanged for the past three decades since the original published success rates by the Vasectomy Reversal Study Group (12). VE is associated with a longer time to patency as well with patients taking 2.8 to 6.6 months to have sperm return to ejaculate as compared to 1.7 to 4.3 months for those undergoing VV. Additionally, of those patients who successfully have sperm return to the ejaculate after VE up to 50% will have delayed failure compared to 12% for those undergoing VV who are patent. Finally, of those who experience delayed failure after undergoing VE it usually occurs earlier with studies reporting as early as 6 months post-operatively (20). Given the lack of improvement and significantly worsened outcomes with VE further surgical refinement is a constant goal for surgeons performing this procedure. Conclusion: In conclusion, this video is both a demonstration and a call to action for commitment to surgical innovation. We aim to raise the bar in VE success rates, ultimately bringing tangible benefits to patients and contributing to the ongoing evolution of reproductive medicine. The novel epididymal occlusion stitch emerges as a beacon of progress, promising not only enhanced safety but also potential reductions in patency time. Surgical excellence and methodological refinement, as exemplified in this video, lay the foundation for a future where male reproductive surgery continues to break new ground. 40 1, 1 (1 2. 2 . 2) 3. (3) 4. 4 (4) vasectomy infections trauma 5. 5 (5) fertility options IVF (IVF) costeffective cost effective 6, (6 7. 7 7) However 99 (VV 65 (VE 8, 8 (8 9. 9 9) Thus 39yearold yearold 39 year old 37yearsold yearsold 37 years cycles levels aspiration right transected obstruction 100 10 0 10- placed incised 70 7- left 3month month 18cc, 18cc cc , (18cc) discomfort postsurgery, postsurgery post surgery, Encouragingly followup approach 10. (10) total viable Historically th century 11. 11 (11) Notably likelihood 199 8. (8) literature 12. 12 (12) 28 66 6. 17 1. 43 Additionally 50 patent Finally postoperatively operatively 20. 20 (20) Conclusion conclusion innovation medicine progress ground ( (3 (4 (5 (IVF (18cc (10 (11 19 (12 (20 (2

ABSTRACT Purpose Varicocele is a condition known to cause damage to seminal parameters and sperm function. Furthermore, it has been hypothesized that the varicocele effect on fertility is time-dependent; however, little is known about the consequences of its establishment time on reproductive organs and/or sperm function. This study aimed to evaluate the effect of the duration of experimental varicocele on reproductive organs, sperm parameters, and sperm function. Materials and Methods Varicocele induction surgeries were performed in Wistar rats aged 40 or 100 days old. At 160-day-old, analyses were performed, including biometry of reproductive organs (prostate, seminal vesicles, epididymis, and testis), sperm parameters (vitality, morphology, and motility), and sperm function tests (nuclear DNA integrity, acrosome integrity, and mitochondrial activity). Results The analysis of the biometry of reproductive organs showed no differences between distinct ages in which varicocele was induced. The total abnormal sperm morphology was bigger in animals with varicocele induced to 100 days old than in animals with varicocele induced to 40 days old. Regarding nuclear DNA integrity, animals of varicocele induced to 100 days old showed worse results compared to animals of varicocele induced to 40 days old. Other parameters analyzed showed no differences between varicocele groups. Conclusion In this study conducted on rats, we conclude that varicocele adversely affects sperm, particularly its function. However, we did not observe a negative progressive effect on sperm. Furthermore timedependent dependent time-dependent however andor 4 10 160dayold, 160dayold dayold 160 day old, 160-day-old prostate, prostate (prostate vesicles epididymis testis, testis , testis) vitality, vitality (vitality motility, motility motility) integrity activity. activity . activity) groups However 1 16

Resumen El objetivo fue determinar si la presencia de cuernos en carneros define si son de jerarquía alta o baja. Se evaluaron 20 carneros Dorper para determinar su rango social (SR), alto (HSR) o bajo (LSR) en condiciones de manejo intensivo. Al momento de la clasificación del SR, se registraron la presencia (HP) o ausencia (HA) de cuernos y distancia entre los mismos, la altura a la cruz, largo del cuerpo, perímetro torácico, circunferencia escrotal, olor, peso corporal y condición corporal. Posteriormente, se realizó la evaluación seminal, registrando el volumen del eyaculado, la concentración espermática y la motilidad masal. No se presentaron diferencias (p > 0.05) para las variables morfométricas entre los grupos. Sin embargo, la HP así como la calidad seminal, fueron mayores (p < 0.05) en los machos HSR. La presencia de cuernos en los carneros determinó su rango social y los machos dominantes mostraron una mejor calidad seminal que los machos subordinados.

Abstract The aim was to determine if the presence of horns on rams defines whether they are high-ranking or low-ranking. Dorper rams (n = 20) were evaluated to determine their social rank (SR), either high (HSR) or low (LSR), under intensive management conditions. Upon SR classification, the morphometric variables horns presence (HP) or absence (HA) and distance between them, the height at the withers, body length, thoracic perimeter, and scrotal circumference, male odor, body weight, and body condition score were registered. Afterward, semen evaluations were performed, recording the ejaculated volume, sperm concentration, and mass motility. There were no differences (p > 0.05) for the morphometric variables between the groups. However, HP as well as semen quality were higher (p < 0.05) in HSR males. The presence of horns in the rams determined their social rank and the dominant males showed better semen quality than the subordinate males.

Abstract In the context of the current debates about the disclosure of donor conception, this paper explores the perspectives on identity of people who have resorted to donating sperm and/or eggs in Buenos Aires City and for whom filiation with their descendants is not based on sharing biogenetic substance. Through the examination of a corpus of interviews conducted between 2018 and 2020 and in dialogue with kinships studies in anthropology, two notions of identity are recognized, identity as a construction or process and identity as an attribute transmitted through genes. Fathers and mothers’ reflections about the identity of their children and the identity of the donors return us to the problem of the biological and social aspects of kinship and the culture/nature binomial, forcing us to reflect on the distinction and articulation between both domains.

Resumen En el marco de los debates actuales sobre el levantamiento del anonimato de los/as donantes de gametos, este artículo indaga las perspectivas sobre la identidad de personas que han recurrido a la donación de semen y/u óvulos en la Ciudad Autónoma de Buenos Aires y cuyo vínculo de filiación con su descendencia no se basa en compartir una substancia biogenética. A partir del examen de un corpus de entrevistas realizadas entre 2018 y 2020 y en diálogo con la antropología del parentesco, se reconocen dos nociones de identidad en juego, la identidad como construcción o proceso y la identidad como un atributo transmitido a través de los genes. Las reflexiones de los padres y las madres acerca de la identidad de sus hijos/as y la identidad de los/las donantes nos devuelven de lleno al problema de los aspectos biológicos y sociales del parentesco y al binomio cultura/naturaleza, obligando a reflexionar sobre la distinción y articulación entre ambos dominios.

Resumen Antecedentes: El incremento seminal de especies reactivas de oxígeno (ERO) se ha vinculado con la oxidación del ADN y anormalidades en la morfología espermática. La enzima 8-oxoguanina ADN glucosilasa 1 (OGG1) repara la oxidación del ADN. Sin embargo, la presencia del polimorfismo en la OGG1 que involucra cambio de citocina (C) por guanina (G), resultando la sustitución de una cisteína por una serina en el codón 326 (Ser326Cis), ha demostrado disminución en la reparación del ADN oxidado. Estudios del polimorfismo Ser326Cis en hombres españoles y asiáticos con infertilidad demostraron que el alelo G(Cis) incrementa las ERO, impactando en la infertilidad y en la teratozoospermia. Objetivo: Conocer la prevalencia del polimorfismo Ser326Cis de OGG1 en pacientes con teratozoospermia. Método: Se analizaron parámetros espermáticos y el polimorfismo Ser326Cis de OGG1 de 81 muestras de semen con teratozoospermia. Resultados: Los genotipos detectados fueron Ser326Ser(CC) 43%, Ser326Cis(CG) 41% y Cis326Cis(GG) 16%. La frecuencia del alelo G(Cis) fue de 0.4, valor mayor a la frecuencia reportada en las bases de datos disponibles para poblaciones americanas (0.21-0.29), los parámetros espermáticos no se relacionaron con el polimorfismo Ser326Cis. Conclusión: El alelo G(Cis) es un factor que contribuye a la infertilidad.

Abstract Background: The increase in seminal reactive oxygen species (ROS) has been linked to DNA oxidation and abnormalities in sperm morphology. The enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) repairs DNA oxidation. However, the presence of the polymorphism in OGG1 that involves a change of cytokine (C) to guanine (G) resulting in the substitution of a cystine for serine at codon 326 (Ser326Cys) has shown a decrease in the repair of oxidized DNA. Studies of the Ser326Cys polymorphism in Spanish and Asian men with infertility demonstrated that the G(Cys) allele increases ROS, impacting infertility and teratozoospermia. Objective: To know the prevalence of the Ser326Cys polymorphism of OGG1 in patients with teratozoospermia. Method: Sperm parameters and the Ser326Cys polymorphism of OGG1 were analyzed from 81 semen samples. Results: The genotypes detected were Ser326Ser(CC) 43%, Ser326Cys(CG) 41%, and yis326Cys(GG) 16%. The frequency of the G allele (Cys) was 0.4, a value higher than the frequency reported in the databases available for American populations (0.21-0.29), the sperm parameters were not related to the Ser326Cys polymorphism. Conclusion: The G (Cys) allele is a factor that contributes to infertility.

Abstract Increasing availability to assisted reproduction (AR) treatments in Argentina and advances in cryobiology resulted in changes in andrology laboratories. The aim of this study was to evaluate the demand and evolution of seminal variables in the last two decades, characterise the current andrology laboratory, evaluate the impact of the incorporation of quality assurance and the introduction of computer assisted semen analysis (CASA). Data were taken from internal quality control (IQC) monthly means (n=22 528) and professionals in charge of laboratories (n=65) and fertility physicians’ (n=33) surveys. Overall demand decreased significantly while first-time orders increased. Sperm volume and sperm count –androgen dependent parameters– decreased over the years. Strict morphology criteria reduced the percentage of normal results; half of the physicians received results between 0 and 10% and 40% considered that it compromised the clinical value of the variable. The CASA system made it possible to objectify sperm kinetic, increasing the percentage of fast progressives, but few laboratories have incorporated it. Sixty-six percent of physicians resolve severe andrological factor by clinical treatments and 95% use AR techniques in those cases. Semen analysis is mainly performed by specialised biochemists, with low adherence to laboratory automatisation and accreditation, but with participation in external quality assessment programmes. The demand decreased because of the increase in AR treatment. The lower percentage of normal forms compromises their clinical utility.

Resumen La mayor accesibilidad a los tratamientos de reproducción asistida (RA) y los avances de la criobiología produjeron cambios en los laboratorios de andrología. El objetivo de este trabajo fue analizar la demanda y evolución de las variables seminales en las últimas dos décadas, caracterizar el laboratorio andrológico actual, evaluar el impacto de la incorporación del aseguramiento de la calidad y la inclusión de los sistemas computarizados (CASA). Se utilizaron datos de las medias mensuales del control de calidad interno (n=22 528) y encuestas a profesionales de laboratorios andrológicos (n=65) y a médicos especialistas en fertilidad (n=33). La demanda global se redujo significativamente con el aumento de las solicitudes de primera vez. El volumen y recuento, variables dependientes de andrógenos, disminuyeron con los años. El criterio estricto en morfología disminuyó el porcentaje de normales; la mitad de los médicos encuestados recibieron resultados entre 0 y 10% y el 40% consideró que ponía en riesgo el valor clínico de la variable. El sistema CASA permitió objetivar la cinética espermática e incrementar el porcentaje de progresivos rápidos, pero pocos laboratorios lo incorporaron. El 66% de los médicos resuelven el factor andrológico severo por tratamientos clínicos y el 95% utiliza técnicas de RA. El análisis de semen es ejecutado fundamentalmente por bioquímicos especializados, con baja adhesión a la automatización y acreditación del laboratorio, pero con participación en programas de evaluación externa de calidad. La demanda disminuyó como consecuencia del aumento del tratamiento por RA. La reducción del porcentaje de formas normales compromete su utilidad clínica.

Resumo O aumento do acesso aos tratamentos de reprodução assistida (RA) e os avanços na criobiologia levaram a mudanças nos laboratórios de andrologia. O objetivo deste trabalho foi analisar a demanda e a evolução das variáveis de sêmen nas últimas duas décadas, caracterizar o laboratório de andrologia atual, avaliar o impacto da incorporação da garantia da qualidade e a inclusão dos sistemas computadorizados (CASA). Foram utilizados dados das médias mensais do controle de qualidade interno (n= 22 528) e pesquisas a profissionais de laboratórios andrológicos e a médicos especialistas em fertilidade (n=33). A demanda geral diminuiu significativamente com o aumento das solicitações de primeira vez. O volume e a contagem de esperma, parâmetros dependentes de andrógenos, diminuíram ao longo dos anos. O critério morfológico rigoroso diminuiu a porcentagem de normais; metade dos médicos entrevistados recebeu resultados entre 0 e 10% e 40% considerou que isso comprometía o valor clínico do parâmetro. O sistema CASA, permitiu objetivar a cinética espermática e aumentar o percentual de progressões rápidas, mas poucos laboratórios o incorporaram. 66% dos médicos resolvem o fator andrológico grave por tratamentos clínicos e 95% utilizam técnicas de RA nesses casos. A análise do sêmen é realizada principalmente por bioquímicos especializados, com baixa aderência à automação e acreditação laboratorial, mas com participação em programas de avalação externa de qualidade. A demanda diminuiu como consequência do aumento do tratamento por RA. A diminuição em percentagem de formas normais compromete sua utilidade clínica.

ABSTRACT Objective: This retrospective study aimed to evaluate the effectiveness of low-dose prednisone as a rescue therapy for patients with deteriorating semen parameters following vasovasostomy. Materials and Methods: Electronic medical records were queried at the University of Miami with documented CPT code 55400 (Bilateral Vasovasostomy) between January 2016 and April 2023. Records were then reviewed to identify patients who demonstrated ≥50% decrease in semen parameters, specifically sperm concentration, motility and total motile sperm count. Patients who were treated with 6 weeks of low-dose prednisone were identified, and baseline semen parameters and subsequent changes after prednisone therapy were assessed. A Mann-Whitney U Test was used to compare semen parameter changes before and after prednisone. Adverse effects associated with prednisone were monitored. Results: A total of 8 patients were identified with deteriorating semen parameters who were treated with 6 weeks of low-dose prednisone. Following prednisone therapy, all patients demonstrated improvements in total motile sperm count (TMSC), with a median improvement of 6 million. The median relative improvement in TMSC was 433%. Sperm concentration and motility also improved compared to post-operative baseline. No adverse effects were reported during the treatment period. Conclusions: Low-dose prednisone therapy appears to be a safe and effective intervention for managing deteriorating semen parameters following VV. The observed improvements in TMSC suggest the potential of prednisone to rescue patients with delayed failure after VV. Further research with larger sample sizes is warranted to confirm the safety and efficacy of low-dose prednisone as a rescue therapy in this specific patient population. Optimizing VV outcomes is crucial in male infertility, and further exploration of steroid therapy and innovative biotechnologies is warranted. Objective lowdose low dose vasovasostomy Methods 5540 Bilateral Vasovasostomy 201 2023 50 ≥50 assessed MannWhitney Mann Whitney monitored Results TMSC, , (TMSC) million 433 433% postoperative post operative period Conclusions Lowdose Low population infertility 554 20 202 5 ≥5 (TMSC 43 55 2 ≥ 4

Abstract 17. The study aimed to evaluate the Fertilization Rate of four independent in vitro treatments of bovine oocytes with Sexed (SX) and Conventional (Conv) semen, capacitated (B.O and Percoll) using the Swim-Up technique. Cleavage was assessed at 48 hours post-fertilization and on the seventh day of embryonic development. The results showed a cleavage percentage of 59.4% in T1 (Conv. Percoll), 60% in T2 (Conv. B.O), 75% in T3 (SX. Percoll), and 46.6% in T4 (SX. B.O), with a statistically significant difference (p>0.05) observed specifically between T3 and T4. The embryo development rate on the seventh day was 34.4% in T1, 20% in T2, 15% in T3, and 10.51% in T4. In this case, a statistically significant difference was found in the use of conventional medium with Percoll compared to SX B.O medium (p<0.05) (T1 and T4, respectively). The results also showed similar production of morula and early blastocysts and protruded embryos among the four treatments, demonstrating the feasibility of in vitro fertilization (IVF) technique for producing bovine embryos. In conclusion, the preliminary evaluation highlights the significant influence of the sexing method and treatment on semen quality and embryonic development in animal reproduction programs. Although Sexed semen with Percoll shows potential to improve cleavage rates, conventional semen, especially in T1 treatment (Conv. Percoll), exhibits superiority in individual motility and concentration. These findings provide crucial insights for optimizing reproductive programs.

Resumen 13. El objetivo del presente estudio fue evaluar la Tasa de Fecundación in vitro de ovocitos bovinos con semen sexado (SX) y convencional (Conv.) capacitados (B.O y Percoll) con la técnica Swim-Up. Se evaluó el clivaje a las 48 horas post fecundación y al séptimo día del desarrollo embrionario. Los resultados mostraron un porcentaje de clivaje del 59,4% en el T1 (Conv. Percoll), 60% en el T2 (Conv. B.O), 75% en el T3 (SX. Percoll) y 46,6% en el T4 (SX. B.O), mostrando diferencia estadística entre T3 y T4 (p>0,05). La tasa de desarrollo del embrión al séptimo día fue 34,4% T1, 20% en T2, 15% en T3 y 10,51% en T4; en este caso, se encontró diferencia estadísticamente significativa en el uso del medio convencional más Percoll en comparación con el medio de SX B.O (p<0,05) (T1 y T4, respectivamente). Los resultados también mostraron una producción similar de mórulas y blastocitos tempranos y protruidos entre los cuatro tratamientos, y demuestran la viabilidad del desarrollo e implementación de la técnica de fertilización in vitro (FIV) para producir embriones bovinos. Concluyendo la evaluación preliminar, destaca la significativa influencia del método de sexado y del tratamiento en la calidad del semen y desarrollo embrionario en programas de reproducción animal. Aunque el semen sexado con Percoll muestra potencial para mejorar tasas de clivaje, el semen convencional, especialmente en el tratamiento T1 (Conv. Percoll), exhibe una superioridad en motilidad individual y concentración. Estos resultados informan decisiones cruciales para optimizar programas reproductivos.

ABSTRACT: The present study evaluated the cryoprotectant efficacy of dimethylacetamide (DMA) and ethylene glycol in a one-step protocol to freeze boar sperm. The sperm-rich portion of the ejaculates from two boars were collected once a week, for 10 weeks. After collection, the ejaculates were diluted (1:1; v/v) in the cooling extender. After determining their spermatozoa concentration, the ejaculates were pooled with the same number of spermatozoa from each boar and stabilized at 20°C for 120 min. Distinct cryoprotectants were added to the cooling extender at 20 °C, at different concentrations, composing six treatments: 1.25% and 2.5% glycerol (control); 1.25% and 2.5% ethylene glycol; 2.5% and 5.0% DMA. The samples were stored in 0.25 mL straws, containing 35 × 106 spermatozoa. After 90 min at 20 °C, the straws were submitted to a cooling curve until 5 °C (0.3 to 0.5 °C/min) and kept at 5°C for 60 min. Freezing was conducted by placing the straws horizontally 5 cm above the liquid nitrogen for 10 min, followed by immersion on liquid nitrogen. After thawing at 37 °C for 30 seconds, sperm quality was evaluated through a computer-assisted semen analysis system and flow cytometry. Sperm motility was greater (P< 0.05) in treatments with 5.0% and 2.5% DMA (22.2 ± 2.6% and 20.0 ± 2.8%, respectively) than in treatment with 2.5% ethylene glycol (8.2 ± 1.0%). The integrity of the plasma membrane (P = 0.08) and mitochondrial membrane potential (P = 0.27) was similar among the treatments. The treatment with 2.5% ethylene glycol was the least efficient to maintain intact acrosome membrane (P< 0.01). Some kinetics parameters (DAP, DCL, DSL, VAP, VCL, VSL e ALH) were positively affected by 5.0% DMA. The one-step freezing protocol resulted in unsatisfactory boar sperm motility after thawing, regardless of the cryoprotectant. ABSTRACT (DMA onestep one step spermrich rich week 1 weeks collection 11 (1:1 v/v vv v concentration 20C C 12 2 concentrations 125 25 1.25 2.5 control (control) 50 0 5.0 025 0.2 3 9 0.3 03 (0. 05 0. °C/min Cmin 5C 6 seconds computerassisted computer assisted cytometry P< P 0.05 005 22.2 222 22 (22. 26 2.6 200 20. 28 8 2.8% respectively 8.2 82 (8. 1.0%. 1.0% . 1.0%) 0.08 008 08 0.27 027 27 0.01. 001 0.01 01 0.01) DAP, DAP (DAP DCL DSL VAP VCL ALH (1: 1.2 2. (control 5. 02 (0 0.0 00 22. (22 2.8 8. (8 1.0 (1 1. ( (2

RESUMO: O presente estudo objetivou avaliar a eficácia de um protocolo one-step de congelamento do sêmen suíno utilizando dimetilacetamida (DMA) e etilenoglicol como crioprotetores. Durante 10 semanas, a fração rica dos ejaculados de dois machos suínos foram coletados, uma vez por semana. Após a coleta, os ejaculados foram diluídos (1:1; v/v) no diluidor de resfriamento. Após a avaliação da concentração espermática, os ejaculados foram agrupados em um pool com o mesmo número de espermatozoides de cada macho e estabilizados a 20 °C por 120 min. Os criopropetores foram adicionados ao diluidor de congelamento a 20 °C, em diferentes concentrações, compondo seis tratamentos: glicerol (controle), 1,25% e 2,5%; etilenoglicol, 1,5% e 2,5%; e DMA, 2,5% e 5,0%. As amostras foram armazendadas em palhetas de 0,25 mL contendo 35 x 106 espermatozoides. Após 90 min a 20 °C as palhetas foram submetidas a uma curva de resfriamento até 5 °C (0,3 a 0,5 °C/mim) e mantidas a 5 °C por 60 min. O congelamento foi realizado a partir da colocação das palhetas horizontalmente a 5 cm acima do nitrogênio líquido por 10 min, com sua posterior imersão no nitrogênio líquido. Após o descongelamento a 37 °C por 30 segundos a qualidade espermática foi avaliada através de um sistema computadorizado e por citometria de fluxo. A motilidade espermática foi maior (P < 0,05) nos tratamentos com 5,0% e 2,5% DMA (22,2 ± 2,6% e 20,0 ± 2,8%, respectivamente) do que no tratamento com 2,5% etilenoglicol (8,2 ± 1,0%). A integridade da membrana plasmática (P = 0,08) e potencial de membrana mitocondrial (P = 0,27) foi similar entre os tratamentos. O tratamento com 2,5% de etilenoglicol foi menos eficiente em manter membrana acrossomal intacta (P< 0,01). Alguns parâmetros de cinética espermática (DAP, DCL, DSL, VAP, VCL, VSL e ALH) foram afetados positivamente pelo uso de DMA a 5.0%. O protocolo simplificado para congelamento de sêmen suíno resultou em motilidade espermática insatifatória após o descongelamento, independente do crioprotetor utilizado. RESUMO onestep one step (DMA crioprotetores 1 semanas coletados semana coleta 11 (1:1 v/v vv v 2 C 12 concentrações controle, controle , (controle) 125 25 1,25 15 1,5 2,5 50 0 025 0,2 3 9 0,3 03 (0, 05 0, °C/mim Cmim mim 6 fluxo P 0,05 005 5,0 22,2 222 22 (22, 26 2,6 200 20, 28 8 2,8% respectivamente 8,2 82 (8, 1,0%. 1,0% . 1,0%) 0,08 008 08 0,27 027 27 P< 0,01. 001 0,01 01 0,01) DAP, DAP (DAP DCL DSL VAP VCL ALH 5.0% utilizado (1: (controle 1,2 1, 2, 02 (0 0,0 00 5, 22, (22 2,8 8, (8 1,0 5.0 (1 ( (2 5.

ABSTRACT. The aim of this study was to investigate the effectiveness of intratesticular injection (ITI) of sodium chloride (NaCl) at two different concentrations for supressing reproductive development in goat kids at two months of age. The experiment was carried out at the experimental unit of Aydın Adnan Menderes University, Çine Vocational School, located in Çine, Aydın, Turkey. The experimental groups were control (C, n = 9), a single ITI of 20% NaCl (T1, n = 8) or 40% NaCl (T2, n = 8). The growth, reproductive characteristics, and histological appearance of the testis in the kids were examined up to nine months of age in different periods. The live weights, scrotal circumference, semen and sexual behavior characteristics were not different among groups. The histogical findings were observed in both testes at nine months of age. Greater intensity of destruction was observed in the testes of T1 and T2 kids than that of C kids. High levels of tubular degeneration were detected in testicular tissue samples in T1 and T2 groups. Based on the results, however, it was concluded that neither 20 nor 40% concentrations of NaCl did ensure the castration effect in the goat kids. ABSTRACT (ITI (NaCl University School Turkey C, (C 9, 9 , 9) T1, T (T1 8 40 T2, (T2 8. . growth periods weights circumference results however 2 (T 4

Abstract Background Varicocele is an abnormal expansion of the pampininias venous plexus in the scrotum, resulting in impaired sperm production and reduced sperm quality. The exact pathophysiological mechanism leading to varicocele-related infertility has not been fully elucidated. Although treatable, varicocele may lead to male infertility. Objective To investigate the relationship between semen parameters, serum InhB and INSL-3 levels, and the degree of varicocele in male patients. Methods Serum InhB and INSL-3 were detected. To evaluate the relationship between semen parameters and serum InhB and INSL-3 levels. To evaluate the value of semen parameters and serum InhB and INSL-3 levels in distinguishing disease severity in patients with varicocele. Results Serum INSL-3 in patients with varicocele decreased with the severity of the disease. Serum INSL-3 was positively correlated with total sperm count and frequency of normal sperm morphology. There was a weak correlation between serum InhB and semen volume, concentration, and total sperm. Patients with different disease severity were similar within the groups, with partial overlap or similarity between varicocele Grade I and Grade II, and significant differences between Grade III and Grade I and II. Semen volume, concentration, total sperm, normal sperm morphology, and serum InhB and INSL-3 levels could distinguish the degree of varicocele. Conclusion Semen parameters and the combination of serum InhB and INSL-3 levels in patients with varicocele are closely related to the severity of the disease. Serum INSL-3 is expected to be a potential biomarker for early clinical intervention. scrotum quality varicocelerelated elucidated treatable INSL3 INSL 3 INSL- detected morphology volume concentration groups II intervention

Abstract One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos. (IVP biotechnology development divisions hormones proteins factors nutrients PlateletRich Platelet Rich (PRP cells Therefore FBS (FBS IVM, IVM , (IVM) cumulusoocyte cumulus (COCs G 5 PRP) MIV SFB) SFB. . Subsequently bull respectively HSP70 HSP stress except group D7 D expected. expected expected) costeffective cost effective HSP7

Abstract This study aimed to evaluate the efficiency of two or three estrus synchronizations followed by artificial insemination (AI) by laparoscopy with frozen semen in sheep. Santa Inês sheep (n=147) were randomly distributed into two groups: Re-sync - 2FTAI (n=72) and Doppler - 3FTAI (n=75), synchronized with a short protocol associated with GnRH and AI by laparoscopy with frozen semen 50 h after removing the P4 device (D0). Two AIs were performed in the Re-sync group, with the start of the resynchronization protocol on D23 and gestational diagnosis on D30; the non-pregnant females were inseminated again. The sheep were resynchronized (D10) in the Doppler group and the gestation diagnosis was made early at 17 days using Doppler ultrasound. The open females were inseminated again, totaling three inseminations in 42 days. Early gestation diagnoses were confirmed at 30 days. Re-sync (29.16%) and Doppler (21.33%) were similar to each other in terms of cumulative gestation rate (P>0.05). The indicators of the non-gestation diagnosis technique at 17 days were sensitivity of 100%, specificity of 28%, positive predictive value of 21.3%, negative predictive value of 100%, and accuracy of 39.8%. Early diagnosis allowed sheep to be inseminated two to three times in the same period and was effective in diagnosing truly non-pregnant females. Therefore, estrus resynchronization associated with AI by laparoscopy using frozen semen can be applied in sheep, but a higher gestation rate could not be achieved with two or three inseminations at the end of the breeding season. (AI n=147 n147 n 147 (n=147 groups Resync Re sync FTAI n=72 n72 72 (n=72 n=75, n75 n=75 , 75 (n=75) 5 P D0. D0 D . (D0) D2 D30 nonpregnant non pregnant again D10 (D10 1 ultrasound 4 3 29.16% 2916 29 16 (29.16% 21.33% 2133 21 33 (21.33% P>0.05. P005 P>0.05 0 05 (P>0.05) nongestation 100 100% 28 28% 213 21.3% 398 39 8 39.8% Therefore season n=14 n14 14 (n=14 n=7 n7 7 (n=7 (n=75 (D0 D3 D1 (D1 29.16 291 2 (29.16 21.33 (21.33 P00 P>0.0 (P>0.05 10 21.3 39.8 n=1 n1 (n=1 n= (n= (D 29.1 (29.1 (21.3 P0 P>0. (P>0.0 21. 39. (n 29. (29. (21. P>0 (P>0. (29 (21 P> (P>0 (2 (P> ( (P

Resumo Objetivou-se avaliar a eficiência de duas ou três sincronizações de estro seguida da inseminação artificial (IA) por laparoscopia com sêmen congelado em ovinos. As ovelhas Santa Inês (n=147) foram distribuídas aleatoriamente em 2 grupos: Re-sync - 2IATF (n=72) e Doppler - 3IATF (n=75), sincronizadas com protocolo curto associado ao GnRH e IA por laparoscopia com sêmen congelado 50 horas após a retirada do dispositivo P4 (D0). No grupo Re-sync foram realizadas duas IA com início do protocolo de ressincronização em D23 e diagnóstico gestacional em D30, as fêmeas não gestantes eram inseminadas novamente. No grupo Doppler as ovelhas foram ressincronizadas (D10) e o diagnóstico de gestação realizado precocemente aos 17 dias, com auxílio da ultrassonografa Doppler. As fêmeas vazias eram inseminadas novamente, totalizando três inseminações em 42 dias. Os diagnósticos precoces de gestação foram confirmados com 30 dias. Re-sync (29,16%) e Doppler (21,33%), foram semelhantes entre si na taxa de prenhez acumulativa (P>0,05). Os indicadores da técnica de diagnóstico de não-gestação aos 17 dias foram: sensibilidade 100%, especificidade 28%, valor preditivo positivo 21,3%, valor preditivo negativo 100% e acurácia 39,8%. Com o auxílio do diagnóstico precoce as ovelhas puderam ser inseminadas de duas a três vezes no mesmo período e mostrou efetividade em diagnosticar as fêmeas verdadeiramente não gestante. Conclui-se que a ressincronização de estro associada a IA por laparoscopia utilizando sêmen congelado pode ser aplicada em ovinos, entretanto não foi possível alcançar maior taxa de prenhez com duas ou três inseminações ao final da estação de monta. Objetivouse Objetivou se (IA ovinos n=147 n147 n 147 (n=147 grupos Resync Re sync IATF n=72 n72 72 (n=72 n=75, n75 n=75 , 75 (n=75) 5 P D0. D0 D . (D0) D2 D30 novamente D10 (D10 1 4 3 29,16% 2916 29 16 (29,16% 21,33%, 2133 21,33% 21 33 (21,33%) P>0,05. P005 P>0,05 0 05 (P>0,05) nãogestação 100 28 28% 213 21,3% 398 39 8 39,8% gestante Concluise Conclui monta n=14 n14 14 (n=14 n=7 n7 7 (n=7 (n=75 (D0 D3 D1 (D1 29,16 291 (29,16 21,33 (21,33% P00 P>0,0 (P>0,05 10 21,3 39,8 n=1 n1 (n=1 n= (n= (D 29,1 (29,1 (21,33 P0 P>0, (P>0,0 21, 39, (n 29, (29, (21,3 P>0 (P>0, (29 (21, P> (P>0 (2 (21 (P> ( (P

Abstract This study aimed to analyze the characteristics of the HSP70 gene and protein in spermatozoa of Bali bulls of different age groups and to examine its potential as a biomarker determining bull fertility. This study used frozen semen produced from six Bali bulls divided into two groups based on age (≤ 9 years and ≥ 12 years). Parameters of frozen semen quality analyzed included sperm motility and kinetics using computer-assisted semen analysis, sperm morphological defects using Diff-Quick staining, acrosome integrity using FITC-PNA staining, and DNA fragmentation using acridine orange staining. HSP70 gene expression characterization was analyzed using qRT-PCR, and HSP70 protein abundance was analyzed using enzyme immunoassays. Fertility field data were obtained by analyzing the percentage conception rate for each bull based on the artificial insemination service data contained in the Indonesian-integrated system of the National Animal Health Information System (iSIKHNAS). The results showed significant differences (P<0.05) in total and progressive motility, morphological defects of the neck and midpiece, and tail of sperm, and acrosome integrity between the age groups of Bali bulls. HSP70 gene expression and protein abundance showed no significant differences (P>0.05) in different age groups. HSP70 gene expression correlated with fertility rate (P<0.05). Age affected several semen quality parameters but did not affect HSP70 gene expression and protein abundance. The HSP70 gene molecule could be a biomarker that determines the fertility of Bali bulls. HSP HSP7 ≤ ( 1 years. . years) computerassisted computer assisted analysis DiffQuick Diff Quick staining FITCPNA FITC PNA qRTPCR, qRTPCR qRT PCR, PCR qRT-PCR immunoassays Indonesianintegrated Indonesian integrated iSIKHNAS. iSIKHNAS (iSIKHNAS) P<0.05 P005 P 0 05 (P<0.05 midpiece P>0.05 (P>0.05 P<0.05. (iSIKHNAS P<0.0 P00 (P<0.0 P>0.0 (P>0.0 P<0. P0 (P<0. P>0. (P>0. P<0 (P<0 P>0 (P>0 P< (P< P> (P> (P