EFFECTS OF BEEF SEMEN AND BEEF EMBRYO STRATEGIES ON PROFITABILITY: Economics of using beef semen and beef in vitro produced embryo transfer in Jersey herds.

Dairy herds have adopted sexed semen (SS) and beef semen (BS) to control heifer inventory and increase calf sales revenue. Beef in vitro-produced embryo transfer (beef IVP-ET) may be an alternative to increase calf sales revenue. Besides, raising those Jersey beef crossbred and/or pure beef animals in a dairy system may be a new source of revenue. We aimed to evaluate breed strategies combining dairy conventional semen (CS), SS, BS, and beef IVP-ET on herd dynamics and profitability by marketing those animals with one-day-old or raising them to 180 kg. A Markov chain model was developed to maximize the profitability of Jersey herds by changing the number of dairy heifers sold at birth and the culling rate of 3rd and greater parity cows. The model presents inputs on the reproductive and productive performance of heifers and cows over time. The last year's data (year 10 - steady state) was used to calculate accrual operational cost and revenue per cow per year. We varied the breeding strategy by breeding order and parities, the embryo transfer cost ($85 or $170), the pure beef calf market price ($200 or $300), and by marketing Jersey-beef and pure beef animals with one-day-old or raising them to 180 kg. A total of 8 scenarios + default scenario were simulated. Overall, the proportion of SS use was 47.3 ± 0.6%. For the scenarios replacing all CS breedings with BS breedings, the proportion of CS and BS used was 52.3 ± 0.6. When beef IVP-ET was used, the percentage of BS and beef IVP-ET used was 22.4 ± 0.1% and 31.0 ± 0.1%, respectively. We observed that when we compared SS:BS with the default scenario, the production of purebred Jersey male calves was reduced by 83.5%, and profit/cow per year was increased from $113.5 to $203.3 with SS:BS. When a beef IVP-ET of $85 per transfer was used (scenarios 2 and 3), profit/cow per year was $145.5 and $176.2 for a pure-beef calf price of $200 and $300, respectively. In scenario 4, with a beef IVP-ET cost of $170, the lowest profit ($52.9 per cow per year) was found when marketing one-day-old pure-beef calves at $200. The highest profit was achieved for scenario raising the Jersey-beef crossbred animals to 180 kg ($232.9, scenario 6), followed by scenario 7 ($222.9, SS:BS:IVP-ET) with an embryo transfer cost of $85. Under the current market conditions, combining SS and BS in the reproductive program was a feasible economic opportunity for Jersey herds, yielding the highest net return. The adoption of beef IVP-ET in a reproductive program can potentially increase profit/cow per year, but its profitability will depend on the beef IVP-ET pregnancy cost, the pure-beef market price, calf performance, and the herd reproductive performance. In conclusion, raising the Jersey-beef crossbred calves may be a profitable strategy, and dairy producers need to evaluate the best option to invest in since it will take an extra risk to produce high-quality animals to the market.

Impact of heat waves on semen quality: A retrospective study in Argentina between 2005 and 2023.

Heat waves, defined as periods with daily temperatures surpassing the historical average for a specific region, have become more frequent worldwide in recent years. Previous studies have reported a negative association between temperature and semen quality, but the focus has mainly been on Asian and European populations. The study included 54,926 men (18-60 years) undergoing routine semen analysis between 2005 and 2023 at CEUSA-LAEH andrology unit, in Buenos Aires, Argentina. Hourly temperature readings were provided by the Servicio Meteorológico Nacional. R programming (R Studio v2022.07.2) was used to define heat waves, calculate key characteristics, visualize results, and perform statistical tests at the IBYME laboratory. During the period studied, a total of 124 days had heat waves (defined after at least 3 consecutive days with 32.3 °C and 22 °C). Men exposed to heat waves during spermatogenesis exhibited lower sperm number (concentration and count; P < 0.0001) and decreased normal morphology (percentage of normal sperm and normal motile count; P < 0.05) compared to those not exposed. These differences were most pronounced between semen samples from years with several heat waves (2013, 2023) and none (2005, 2007, 2016), displaying 4-5 times higher fold changes (P < 0.05). Further analysis employing multiple regression revealed a significantly negative association between semen quality and heat wave length, suggesting that a prolonged exposure may be more detrimental than an acute exposure. Subsequent analysis focusing on prolonged exposure (≥6-days heat wave) during spermatogenesis revealed a negative (P < 0.05) association between early exposure (spermatocytogenesis: 64-90 days prior semen collection) and semen quality. This study underscores the negative association between early exposure to heat waves during sperm development and semen quality, raising concerns about its possible association with the worldwide declining male fertility. A comprehensive collaborative approach is crucial, involving global governmental policies, sustainable practices, and coordinated efforts across scientific, healthcare, and policy domains.

Timing of semen cryopreservation: before or after processing?

Objective: Seminal cryopreservation causes significant damage to the sperm; therefore, different methods of cryopreservation have been studied. The aim of the study was to compare the effects of density gradient processing and washing/centrifugation with seminal plasma removal for cryopreservation in semen parameters. Methods: Seminal samples of 26 normozoospermic patients were divided into 3 parts: with seminal plasma; after washing/centrifugation; and after selection through density gradient. The samples were cryopreserved for at least two weeks. Motility, sperm count, morphology and viability were evaluated before cryopreservation and after thawing. Results: Density gradient processing selected motile and viable sperm with normal morphology in fresh samples (p<0.05). Cryopreservation negatively affected all sperm parameters regardless of the processing performed, and even if the sperm recovery was lower in the density gradient after the thawing, progressive motility, total motility, viability and morphology remained higher (p<0.05). Conclusion: Cryopreservation significantly compromises sperm parameters (motility, morphology, viability). In normozoospermic patients, the density gradients select better quality spermatozoa compared to other processing methods; this benefit was kept after thawing.

High percentage of midpiece defects in Brangus bull sperm with no reduction in sperm kinematics.

The study investigated midpiece defects in sperm from a 5-year-old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16-month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization.

Cryopreservation of spermatozoa from black-and-gold howler monkeys (Alouatta caraya) using egg yolk-based or soy lecithin-based extenders.

There are more than 200 species and subspecies of Neotropical Primates of which more than 40% are listed as threatened by the IUCN Red List of Threatened Species. Both in situ and ex situ conservation programs can benefit from the use of assisted reproductive technologies. The objective of this study was to evaluate, for the first time, cryopreservation techniques for Alouatta caraya semen. Semen samples were collected from five adult males, analyzed, and frozen in either Test-egg yolk or Test-soy lecithin-based extenders containing either 3 or 4% glycerol. Frozen-thawed samples were analyzed at 10, 40, and 80 min post-thaw. Egg yolk-based extenders were overall better than soy lecithin-based extenders. There was no significant difference between 3 and 4% glycerol in any of the parameters analyzed, however, 4% glycerol in egg yolk-based extender produced more favorable results for total motility, intact plasma membrane, lipid peroxidation, and DNA fragmentation index. This study brought novel information on semen characteristics and cryopreservation aspects for A. caraya, which can help shape future experiments to improve the outcome of frozen-thawed sperm for this and other species of Neotropical primates.

Sperm HSP70: may not be an age-dependent gene but is associated with field fertility in Bali bulls (Bos sondaicus).

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.

Abusive use of anabolic androgenic steroids, male sexual dysfunction and infertility: an updated review.

The evolving prevalence of anabolic androgenic steroids (AAS) abuse among nonathletes is alarming because of the known harm to an individual's health. Among the adverse effects of AAS abuse, male infertility and sexual dysfunction have been often reported in the literature, but little is known regarding its actual prevalence, possible underpinning mechanisms, and potential treatments either during or post-AAS usage. Thus, the current narrative review summarizes the state-of-art regarding the effects of AAS on male fertility and sexual function. Evidence was gathered from the latest reviews and recent original studies, specifically from prospective cohorts and clinical trials, ultimately resulting in five main topics of discussion. First, AAS usage is briefly characterized by its historical background, main physiological mechanisms, and the most frequently used AAS substances. Second, data on the prevalence of AAS-induced male infertility and sexual dysfunction are described. Third, some new insights on possible underpinning mechanisms of AAS-induced male infertility and sexual dysfunction are thoroughly discussed, with particular attention to histological data derived from animal models and the latest insights from prospective cohorts in humans. Fourth, the potential treatments during and after the AAS usage are presented, highlighting the odds of resolving male infertility and sexual dysfunction. Fifth, future directions on this topic are discussed, focusing on the methodological robustness of scientific studies.

Dataset of seminal plasma proteome of Nellore bulls with high and low percentage of abnormalities sperm.

OBJECTIVES: Bovine seminal plasma proteins perform several functions related to sperm function. Changes in the expression pattern or abundance of seminal proteins are related to changes in the fertilizing capacity of bulls. Considering the role of seminal plasma proteins in sperm function and animal reproduction, we investigated changes in the protein abundance profile in response to sperm morphological changes using a proteomic approach. DATADESCRIPTION: In our present investigation, we employed liquid chromatography coupled with mass spectrometry to elucidate the proteomic composition of seminal plasma obtained from Nellore bulls exhibiting varying percentages of sperm abnormalities. Following semen collection, seminal plasma was promptly isolated from sperm, and proteins were subsequently precipitated, enzymatically digested using porcine trypsin, and subjected to analysis utilizing the Acquity nano UHPLC System in conjunction with a mass spectrometer. This dataset encompasses a total of 297 proteins, marking the inaugural instance in which a comparative profile of seminal plasma proteins in young Nellore bulls, categorized by their sperm abnormality percentages, has been delineated using LC-MS/MS. The comprehensive nature of this dataset contributes pivotal proteomic insights, representing a noteworthy advancement in our understanding of the reproductive biology of the Nellore breed.

Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions.

BACKGROUND: Artificial insemination with cooled-shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions. OBJECTIVE: To assess sperm quality parameters and fertility of cooled-stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders. STUDY DESIGN: Controlled and field study. METHODS: In Experiment 1, semen was collected from 21 stallions classified as having good ('Good-coolers', n = 8) or poor ('Bad-coolers', n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk-based (SM) diluent, and refrigerated for 24 h. Then, the cooled-stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM-P), or an egg yolk-based (EY) extender. Unprocessed cooled-stored semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. Experiment 2, cooled semen from 9 stallions classified as Bad-coolers was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM-P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression. RESULTS: Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM-P (p < 0.0001). Semen processed by SpermFilter® resuspended in SM-P was similar to EY (p > 0.05). Pellet resuspension with EY and SM-P improved the HMMP of Bad-cooler stallions (p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen (p < 0.0001). Mares inseminated with SpermFilter®/SM-P (50%, 8/16) or SpermFilter®/-EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) (p < 0.001). MAIN LIMITATIONS: Low number of mares in the fertility trial. CONCLUSION: Sperm quality and fertility of Bad-cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM-P.

Use of thermography in the long-term evaluation of scrotal surface temperature and its impact on seminal quality in stallions.

Scrotal surface thermography is a non-invasive method for assessing testicular thermoregulation in stallions; however, few studies have explored the application of this technique concerning the thermal physiology of equine reproductive systems. This study aimed to evaluate the consistency of testicular thermoregulation in stallions over a year using thermography to measure the scrotal surface temperature (SST). Moreover, we assessed the best region for measuring the surface body temperature compared with the SST. Ten light-breed stallions were used in the experiment. Thermographic images of the scrotal and body surfaces (neck and abdomen) were captured. Fresh, cooled and frozen-thawed semen samples were evaluated to verify the impact of thermoregulation on semen quality. Testicular thermoregulation was maintained throughout the year in stallions amidst changes in the external temperature, as evidenced by the weak correlation between the SST and ambient temperature. A lower correlation was observed between the environmental temperature and body surface temperature (BTS) obtained from the abdomen (BTS-A; R = .4772; p < .0001) than with that obtained from the neck (BTS-N; R = .7259; p < .0001). Moreover, both BTS-A and SST were simultaneously captured in a single image. The consistent quality of the fresh, cooled and frozen semen suggests efficient thermoregulation in stallions throughout the year.

Comparison of different cryoprotectants for freezing donkey (Equus asinus) semen.

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7 %) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n = 8; r = 3) were divided into 4 extenders: BotuSemen Gold with 5 % or 7 % MF and 5 % or 7 % DMF, all containing 11 % lactose, 20 % egg-yolk and 0.5 % Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P > 0.05). However, samples with 5 % DMF showed the highest percentages of sperm with acrosomes and functional membranes (DMF: 5 %: 53.67 ± 22.01; 7 %: 33.92 ± 23.4; MF: 5 %: 44.5 ± 20.46; 7 %: 38.75 ± 27.4) (Data: mean ± SD; P > 0.05). Hence, thirty mares were inseminated: 15 with 5 % DMF and 15 with 7 % DMF. The pregnancy rate was 46 % (7/15) and 0 % (0/15) using the extender with 5 % or 7 % DMF, respectively (P = 0.003). To conclude, the use of 5 % or 7 % of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5 % DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates.

Potential of semen coicis in enhancing the anti-tumor effects of PD-1 inhibitor on A549 cell lines by blocking the PI3K-AKT-mTOR pathway.

BACKGROUND: The objective of this research was to investigate how the combination of semen coicis extract and PD-1 inhibitors can potentially work together to enhance the anti-tumor effects, with a focus on understanding the underlying mechanism. METHODS: We obtained the active components and specific targets of semen coicis in the treatment of NSCLC from various databases, namely TCMSP, GeneCard, and OMIM. By utilizing the STRING database and Cytoscape software, we established a protein interaction network (PPI) for the active ingredient of semen coicis and the target genes related to NSCLC. To explore the potential pathways involved, we conducted gene ontology (GO) and biological pathway (KEGG) enrichment analyses, which were further supported by molecular docking technology. Additionally, we conducted cyto-inhibition experiments to verify the inhibitory effects of semen coicis alone or in combination with a PD-1 inhibitor on A549 cells, along with examining the associated pathways. Furthermore, we investigated the synergistic mechanism of these two drugs through cytokine release experiments and the PD-L1 expression study on A549 cells. RESULTS: Semen coicis contains two main active components, Omaine and (S)-4-Nonanolide. Its primary targets include PIK3R1, PIK3CD, PIK3CA, AKT2, and mTOR. Molecular docking experiments confirmed that these ingredients and targets form stable bonds. In vitro experiments showed that semen coicis demonstrates inhibitory effects against A549 cells, and this effect was further enhanced when combined with PD-1 inhibitors. PCR and WB analysis confirmed that the inhibition of the PI3K-AKT-mTOR pathway may contribute to this effect. Additionally, semen coicis was observed to decrease the levels of IFN-γ, IL-6, and TNF-α, promoting the recovery of the human anti-tumor immune response. And semen coicis could inhibit the induced expression of PD­L1 of A549 cells stimulated by IFN­Î³ as well. CONCLUSION: Semen coicis not only has the ability to kill tumor cells directly but also alleviates the immunosuppression found in the tumor microenvironment. Additionally, it collaboratively enhances the effectiveness of PD-1 inhibitors against tumors by blocking the activation of PI3K-AKT-mTOR.

Transmission electron microscopy reveals the presence of SARS-CoV-2 in human spermatozoa associated with an ETosis-like response.

BACKGROUND: Severe acute syndrome coronavirus 2 can invade a variety of tissues, including the testis. Even though this virus is scarcely found in human semen polymerase chain reaction tests, autopsy studies confirm the viral presence in all testicular cell types, including spermatozoa and spermatids. OBJECTIVE: To investigate whether the severe acute syndrome coronavirus 2 is present inside the spermatozoa of negative polymerase chain reaction-infected men up to 3 months after hospital discharge. MATERIALS AND METHODS: This cross-sectional study included 13 confirmed moderate-to-severe COVID-19 patients enrolled 30-90 days after the diagnosis. Semen samples were obtained and examined with real-time polymerase chain reaction for RNA detection and by transmission electron microscopy. RESULTS: In moderate-to-severe clinical scenarios, we identified the severe acute syndrome coronavirus 2 inside spermatozoa in nine of 13 patients up to 90 days after discharge from the hospital. Moreover, some DNA-based extracellular traps were reported in all studied specimens. DISCUSSION AND CONCLUSION: Although severe acute syndrome coronavirus 2 was not present in the infected men's semen, it was intracellularly present in the spermatozoa till 3 months after hospital discharge. The Electron microscopy (EM) findings also suggest that spermatozoa produce nuclear DNA-based extracellular traps, probably in a cell-free DNA-dependent manner, similar to those previously described in the systemic inflammatory response to COVID-19. In moderate-to-severe cases, the blood-testes barrier grants little defence against different pathogenic viruses, including the severe acute syndrome coronavirus 2. The virus could also use the epididymis as a post-testicular route to bind and fuse to the mature spermatozoon and possibly accomplish the reverse transcription of the single-stranded viral RNA into proviral DNA. These mechanisms can elicit extracellular cell-free DNA formation. The potential implications of our findings for assisted conception must be addressed, and the evolutionary history of DNA-based extracellular traps as preserved ammunition in animals' innate defence might improve our understanding of the severe acute syndrome coronavirus 2 pathophysiology in the testis and spermatozoa.

Effect of environmental factors on seminal microbiome and impact on sperm quality.

Objective: This review provides a comprehensive overview of the existing research on the seminal microbiome and its association with male infertility, while also highlighting areas that warrant further investigation. Methods: A narrative review was conducted, encompassing all relevant studies published between 1980-2023 on the male reproductive tract microbiome in humans. This review considered studies utilizing culture-based, polymerase chain reaction (PCR)-based, and next-generation sequencing (NGS)-based methodologies to analyze the microbiome. Data extraction encompassed sample types (semen or testicular tissue), study designs, participant characteristics, employed techniques, and critical findings. Results: We included 37 studies comprising 9,310 participants. Among these, 16 studies used culture-based methods, 16 utilized NGS, and five employed a combination of methods for microorganism identification. Notably, none of the studies assessed fungi or viruses. All NGS-based studies identified the presence of bacteria in all semen samples. Two notable characteristics of the seminal microbiome were observed: substantial variability in species composition among individuals and the formation of microbial communities with a dominant species. Studies examining the testicular microbiome revealed that the testicular compartment is not sterile. Interestingly, sexually active couples shared 56% of predominant genera, and among couples with positive cultures in both partners, 61% of them shared at least one genital pathogen. In couples with infertility of known causes, there was an overlap in bacterial composition between the seminal and vaginal microbiomes, featuring an increased prevalence of Staphylococcus and Streptococcus genera. Furthermore, the seminal microbiome had discernible effects on reproductive outcomes. However, bacteria in IVF culture media did not seem to impact pregnancy rates. Conclusion: Existing literature underscores that various genera of bacteria colonize the male reproductive tract. These organisms do not exist independently; instead, they play a pivotal role in regulating functions and maintaining hemostasis. Future research should prioritize longitudinal and prospective studies and investigations into the influence of infertility causes and commonly prescribed medication to enhance our understanding of the seminal microbiota's role in reproductive health.

Comparison of SPAG11A gene expression in infertile men with grade 1 and 2 varicocele before and after treatment.

OBJECTIVE: Sperm Associated Antigen 11A (SPAG11A) protein is a family of the epididymis-specific secretory proteins implicated in sperm maturation and function. Varicocele might cause pathophysiological difficulties in the testis and epididymis, with a harmful effect on the environment for spermatogenesis and sperm maturation. The aim of this study was to evaluate the expression level of the SPAG11A gene and sperm parameters in infertile men with grade 1 and 2 varicocele before and after treatment. METHODS: Semen specimens were collected from 20 infertile men with varicocele pre-and post-treatment and 10 healthy volunteers. Semen analysis was conducted according to world health organization guidelines. Real time PCR (qRT-PCR) reaction was applied for determination of SPAG11A mRNA expression. RESULTS: The results showed that there was a significant difference between the concentration and normal morphology between pre- and post-treatment groups and the controls. There were significant differences between pre-treatment and control groups in terms of progressive and non-progressive mobility. SPAG11A mRNA levels were significantly lower in the pre-treatment group than in healthy control subjects (p=0.007). There was no statistically significant difference in the expression of SPAG11A as well as semen parameters in the post-treatment group compared to the pre-treatment group. CONCLUSIONS: SPAG11A gene expression and semen parameters may be affected by varicocele. Whether varicocele treatment is an effective approach to reduce the adverse effect of this disease on SPAG11A expression and semen parameters needs further investigation.

Effect of Diluents and Storage Time on the Cryopreservation of Collared Peccary (Pecari tajacu) Semen after Cooling Storage in a Transport Container at 5 °C.

We verified the possibility of cooling peccary semen for 4, 24, and 48 h before cryopreservation, using different dilution media (TRIS + egg yolk (20%) and PRIMXcell Ultra). Ten ejaculates were divided equally into six aliquots and then diluted. Two aliquots were stored in a biological incubator (4 h), and the remaining aliquots were stored in a commercial container, the Botutainer® (24 and 48 h), both at 5 °C. The samples were cryopreserved and then evaluated for kinetic parameters, functionality, integrity, mitochondrial activity, morphology, and sperm binding capacity. After thawing, samples diluted in TRIS showed total motility of 43.4 ± 6.8%, 48.4 ± 6.2%, and 38.6 ± 5.0% after cooling for 4, 24, and 48 h before cryopreservation, respectively. Such results are significantly greater than those achieved with the use of PRIMXcell diluent for 4 (8.3 ± 2.8%), 24 (4.7 ± 1.4%), and 48 h (4.8 ± 2.9%) storage (p < 0.05). Furthermore, TRIS provided better preservation of sperm membrane integrity when samples were cooled for 24 h (44.5 ± 4.7%) before cryopreservation compared to those samples diluted in PRIMXcell Ultra stored for 24 (25.7 ± 4.0%) and 48 h (25.2 ± 4.0%) before freezing (p < 0.05). In summary, we suggest TRIS diluent + egg yolk (20%) as an effective option to allow semen to cool for 24 or 48 h in a transport container before cryopreservation.

El laboratorio de fertilidad masculina, su evolución en los últimos 20 años / The male fertility laboratory, its evolution over the last 20 years / O laboratório de fertilidade masculina, sua evolução ao longo dos últimos 20 anos

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.

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.

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.

Relationship between semen parameters, serum InhB, and INSL-3 levels, and the degree of varicocele.

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.

Evaluation of pharmacological alternatives to reduce the pain and discomfort produced by electroejaculation in rams.

Electroejaculation (EE) represents the main technique for semen collection from domestic and wild animals independently of libido. However, the technique is associated with intense involuntary muscle contractions, vocalization, ataxia and lying down, caused by the electric stimulation of the nerves in the caudal epigastric region. These clinical manifestations represent important indicators of discomfort. In this context, the objective of this study was to evaluate two protocols of local anaesthetic blockade and two anatomical access for pharmacological desensitization of the caudal epigastric innervation as alternatives to promote comfort and reduce stress associated with EE in rams. For the study, four clinically healthy Dorper rams were selected. All animals were subjected to a design consisting of five semen collection treatments (n = 3 collections per treatment): T1-control, conventional EE without local anaesthetic blockade; T2, EE with ventral blockade (VB) of epigastric innervation using lidocaine hydrochloride 2%; T3, EE with VB of epigastric innervation using a combination of lidocaine hydrochloride 2% and fentanyl citrate; T4, EE with blockade of epigastric innervation through the perineal access using lidocaine hydrochloride 2%; T5, EE with blockade of epigastric innervation through the perineal access using a combination of lidocaine hydrochloride and fentanyl citrate. Seminal samples resulting from EE were subjectively evaluated for sperm motility and concentration, vigour and volume. Additionally, blood serum samples were collected for quantification of cortisol and creatine kinase (CK) enzyme. Assessments of stress and discomfort were conducted by measuring blood pressure, heart rate (HR) and respiratory rate (RR), as well as observing involuntary muscle contractions, ataxia and animal vocalization. No variations in blood pressure, sperm motility, vigour, CK, and cortisol were observed among the treatments. Individual variations were observed for the occurrence of vocalization (p = .0066), but there were no differences between the groups. Anaesthetic blockades conducted using the combination of lidocaine and fentanyl resulted in a lower incidence of ataxia during EE (p < .0001). It is concluded that the combination of fentanyl citrate and lidocaine hydrochloride results in less discomfort for animals undergoing EE, regardless of the anatomical access used for local anaesthetic blockades.

Quality and redox state of bovine sperm cryopreserved with resveratrol use of resveratrol in bovine semen.

The use of antioxidants for semen preservation prevents oxidative damage caused by reactive oxygen species (ROS). One of the most promising natural antioxidants is resveratrol, a phytoalexin derived from plants, grapes, berries, peanuts and red wine. To evaluate the effect of resveratrol on the quality and redox status of cryopreserved bovine semen. Five bulls were subjected to electroejaculation to obtain 15 ejaculates. Each ejaculate was extended with a tris-egg yolk-glycerol-based medium and divided into six aliquots supplemented with 0 (control), 10, 20, 30, 40 and 50 µM of resveratrol. Semen was frozen with liquid nitrogen vapours. Post-thawing, motility and kinetics were evaluated using a computer-assisted sperm analysis (CASA) system, membrane integrity using the hypoosmotic test (HOST), morphology by staining with eosin-nigrosin, sperm vitality by fluorescence microscopy with the SYBR14/IP probes. Total antioxidant capacity (TAC) was evaluated using the ABTS•+ assay and ROS was evaluated using spectrofluorimetry with the H2 DCFDA probe. For the statistical analysis linear models were adjusted and means were compared using the Tukey test. All concentrations of resveratrol reduced post-thawed motility and kinetics of sperm. Supplementation with 40 and 50 µM of resveratrol reduced sperm kinetics, and between 30 and 50 µM of resveratrol alterations in the sperm membrane and morphology were observed. However, using resveratrol at 50 µM increased TAC and at 20 µM, it reduced ROS production of cryopreserved bovine semen. Resveratrol appears to have a dose-dependent effect in which higher doses produce greater sperm alterations, however, it can increase semen TAC during freezing. It is concluded that resveratrol can increase antioxidant capacity and reduce ROS production in cryopreserved bovine semen. However, its use between 10 and 50 µM reduces post-thawing semen quality.