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.

Low-density lipoproteins, resveratrol and quercetin as alternative additives to improve boar semen cooling.

Low-density lipoproteins (LDL), quercetin (Q) and resveratrol (R) have been used for sperm preservation to improve sperm quality in different species. To evaluate the effects of LDL, Q and R during the cooling of boar semen. Fifteen boar semen samples were diluted in a BTS extender supplemented with the treatments: LDL at 6%, Q at 10 µM (Q10), 30 µM (Q30) and 50 µM (Q50), or R at 10 µM (R10), 30 µM (R30) and 50 µM. A control without supplementation was included. The semen was stored by cooling at 16°C for 96 h. Every 24 h, sperm motility and kinetics were evaluated using a computer-assisted sperm analyzer (IVOS). At 24 and 96 h of cooling, functional membrane integrity and mitochondrial membrane potential (ΔΨM) of sperm were evaluated by the hypoosmotic swelling test (HOST) an flow cytometry with JC-1 probe, respectively, LDL improved progressive motility of sperm during cooling. Likewise, LDL increased average path velocity (VAP) and straight-line velocity (VSL) and/or curvilinear velocity (VCL) during the first 48 h of cooling. The use of Q between 10 and 30 µM caused a reduction in total motility, progressive motility and amplitude of the lateral head displacement during the entire cooling period, as well as a decrease in VAP, VSL and VCL at 96 h of cooling. LDL, Q10, Q30 and Q50 modulated mitochondrial activity by reducing high-ΔΨM sperm at 0 and 96 h of cooling. During the cooling of the boar semen prior to artificial insemination, the parameters of sperm quality that could influence fertility decrease; however, the inclusion of antioxidants and additives that protect the plasma membrane, such as LDL, could mitigate the damaging effects on spermatozoa. It is concluded that LDL can improve the motility and kinetics of boar semen during cooling while it could modulating the sperm's mitochondrial activity. On the contrary, Q could alter the motility and kinetics of boar sperm during the cooling period.

Cooling of porcine semen in an extender supplemented with carvacrol.

The addition of antioxidants in boar semen is an alternative to mitigate the reduction of sperm quality during preservation. To evaluate the effect of carvacrol on cooling of boar semen. Fifteen ejaculates from five boars were extended in MR-A® with 0, 5, 10, 15, 15, 20, 25 and 30 µM of carvacrol (C) and were cooled for 5 days at 16°C. Sperm motility and kinetics were evaluated with computer-assisted semen analysis (CASA). At 0 and 96 h, membrane functionality was determined by hypoosmotic test; reactive oxygen species (ROS) production and total antioxidant capacity (TAC) by spectrofluorimetry and mitochondrial membrane potential (Δ¥M) by flow cytometry. Linear models, regression analysis and comparison of means by Duncan test, were conducted. The addition of carvacrol did not influence sperm motility, but at low concentrations decreased ROS production, whereas 30 µM C reduced the membrane functionality and 25 µM C decreased Δ¥M. In addition, regression coefficients showed that C produced a lower rate of decrease in different parameters of sperm motility and kinetics. During cooling there is a reduction in sperm quality due to the excessive production of ROS, generating oxidative stress and affecting cell permeability and functionality. In this study, it was possible to demonstrate the protective activity of C as a molecule capable of neutralizing free radicals. In addition, it has been proposed that C is also capable of reducing peroxyl radicals, superoxide radicals, hydrogen peroxide and nitric oxide. Carvacrol can mitigate the reduction of boar semen quality during the storage period under cooling conditions. Likewise, it can reduce ROS production and modulate the mitochondrial activity of porcine sperm.

Dry food affects the oxidative/antioxidant profile of dogs.

BACKGROUND: Including adequate concentrations of antioxidants in dog diets has been recommended to reduce their vulnerability to the action of free radicals and reactive oxygen species (ROS). Oxidative stress in dogs has been associated with a wide range of diseases and disorders, as well as with ageing. There are few reports about the influence of diet on dog's antioxidant profile and oxidative stress. OBJECTIVE: The objective of this study was to evaluate the effect of four types of dry dog food on the oxidative/antioxidant profile of dogs. METHODS: Six Beagle dog males were used. The study included four experimental diets (dry foods A-D). Each dry food was supplied for 5 weeks to all dogs, for a total of 24 weeks, including an adaptation week between one food and another. For each dry dog food, the total phenolic content (TPC), total antioxidant capacity (TAC) and cytotoxicity were evaluated. Each week, a blood sample was collected to measure ROS and TAC of plasma. A crossover repeated measures design was used. Mixed models were adjusted, and means were compared using the Tukey test. RESULTS: Food A had the highest values for TPC and TAC. Food C had the lowest levels of ROS, whereas food B had the highest TAC in the blood plasma. The dog had a significant influence on the redox state of its blood plasma, even when the same dog was fed the different dry foods. CONCLUSION: Dry dog food influences the oxidative/antioxidant profile of dog's blood plasma; however, this seems to be unrelated to the antioxidant profile of the food.

Evaluation of the effect of conventional and natural antifungals on motility and kinetics of cooled stallion semen.

Microbial growth in semen may cause a decline of sperm quality and fertility; however, the addition of antifungals to semen extender has been shown to impair the overall fertility of the sperm. The aim of this study was to evaluate the antifungal activity of conventional and natural compounds, and their effect on the motility and kinetics of cooled stallion semen. A total of 15 ejaculates from five stallions were collected using the artificial vagina. Each ejaculate was supplemented with: fluconazole at 12.5 (F1), 25 (F2) and 50 (F3) mg/ml; amphotericin-B at 6.5 (A1), 12.5 (A2) and 25 (A3) mg/ml (A3); clotrimazole at 12.5 (C1), 25 (C2) and 50 (C3) mg/ml; isoespintanol at 50 (I1), 100 (I2) and 150 (I3) µM; thymol at 50 (T1), 100 (T2) and 150 (T3) µM; and a control without supplementation. Motility and kinetics of semen at 0, 24 and 48 hr of cooling at 15°C were assessed using computer-assisted sperm analysis (CASA). At hour 48 of cooling, the antifungal effect of the treatments was evaluated. At hour 0 of cooling, amphotericin-B and I3 showed a reduction in most of the motility and kinetic parameters evaluated (p < .05). These treatments, and also C2 and C3, showed similar results at 24 and 48 hr of cooling. Thymol maintained motility and kinetics of the spermatozoa at all evaluated refrigeration times. Besides, I2 showed a decrease (p < .05) in the colony-forming unit compared to that in the control. It is concluded that thymol and isospintanol could be added as natural antifungals in extenders for stallion semen refrigeration.

Cryoprotective Effects of Ergothioneine and Isoespintanol on Canine Semen.

Sperm undergo oxidative stress due to excessive production of reactive oxygen species (ROS) during cryopreservation. Some unconventional natural antioxidants can reduce ROS-induced changes in cryopreserved canine sperm. This study aimed to identify the cryoprotective effects of ergothioneine and isoespintanol on the quality of thawed canine semen. Twelve ejaculates from six dogs were cryopreserved in a tris-yolk extender without (control) or with 50 (E50), 100 (E100), or 150 (E150) µM ergothioneine or 20 (I20), 40 (I40), or 60 (I60) µM isoespintanol. We evaluated the motility and kinetics of thawed sperm using computerized analysis; determined morphology by eosin-nigrosin staining; functional membrane integrity using hypoosmotic tests, and structural membrane and acrosome integrity; mitochondrial membrane potential by fluorescence microscopy; and ROS production by spectrophotometry. Data were statistically analyzed using mixed models and Tukey tests. E100 increased total (60.6% vs. 49.6%) and progressive (26.4% vs. 20.1%) motility, straight line velocity (41.3 vs. 35.9 µm/s), and rapid sperm (17.6% vs. 12.3%) compared with controls. However, E150 reduced the numbers of hyperactive sperm. E100, I40, and I60 reduced the abnormal morphology and ROS production, and all concentrations of both antioxidants increased acrosomal integrity. We concluded that ergothioneine and isoespintanol reduce deleterious sperm alterations and oxidative stress in thawed canine semen.

Membrane stability and mitochondrial activity of bovine sperm frozen with low-density lipoproteins and trehalose.

Cryopreservation results in the destabilization of the sperm plasma membrane, leading to negative side effects such as premature cryocapacitation, apoptosis and the low mitochondrial activity of bovine spermatozoa. Low-density lipoproteins (LDL) and trehalose have been used in seminal freezing to protect the integrity and stability of sperm membranes. Likewise, trehalose can increase the mitochondrial activity of sperm. The objective of this study was to evaluate the membrane stability and mitochondrial activity of bovine sperm after being frozen and treated with LDL sources and trehalose. Ten ejaculates from five bulls were cryopreserved under the treatments, CEY: chicken egg yolk (20% v/v); CCEY: centrifuged CEY (20% v/v); LDL: LDL (8% v/v); T: trehalose (100 mM); and TLDL: T (100 mM) plus LDL (8% v/v). After thawing, membrane stability and mitochondrial membrane potential (ΔΨM) were assessed by flow cytometry through the M-540/Yopro-1 and DiOC6/PI probes. The structural membrane integrity (SMI) was evaluated by fluorescence microscopy using SYBR14/PI dyes. A generalized linear model was adjusted, and the means were compared using the Tukey test. Centrifuged chicken egg yolk and LDL had a higher proportion of non-cryocapacitated non-apoptotic sperm (M-Y-), while CEY and T had the largest populations of cryocapacitated non-apoptotic sperm (M+Y-) and cryocapacitated apoptotic sperm (M+Y+). Centrifuged chicken egg yolk also showed a higher proportion of sperm with high-ΔΨM. Treatments that included egg yolk or purified LDL had a positive effect on SMI. Centrifuged chicken egg yolk has a superior cryoprotective effect on membrane stability and mitochondrial activity of bovine semen over the conventional use of CEY or the individual or simultaneous use of LDL and trehalose.

Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity.

Maintaining the integrity of equine sperm subjected to preservation protocols is essential for the successful development of assisted reproduction procedures. The aim of this study was to assess the mitochondrial membrane potential, lipid peroxidation, and DNA integrity of equine sperm subjected to freezing, vitrification, and freeze-drying. Eight ejaculates obtained from four Colombian Creole horses were subjected to programmable freezing, vitrification, and freeze-drying. After thawing or rehydration, sperm motility and kinetics were assessed through a CASA system. The mitochondrial membrane potential (ΔΨM), lipid peroxidation (LPO), and DNA fragmentation index (DFI) of the spermatozoa were assessed by flow cytometry using the DiOC6 (3), C11-Bodipy 581/591, and propidium iodide (PI) fluorescent dyes. The statistical analysis was conducted via generalized linear models, mean comparisons via the Duncan test, and a principal component analysis. A higher rate of spermatozoa with a high ΔΨM was found for freeze-drying (40.26 ± 7.79%) compared with freezing (21.82 ± 5.38%) and vitrification (5.32 ± 1.17%) (P < .05). Likewise, a higher rate of nonperoxidized viable spermatozoa (Bodipy-/PI-) was found for freeze-drying (35.98 ± 7.01%) in relation to frozen (10.34 ± 2.69%) and vitrified (7.07 ± 2.00%) sperm (P < .05). The DFI of vitrified spermatozoa (0.12 ± 0.04%) was higher when compared with the frozen (0.03 ± 0.01%) and freeze-dried (0.02 ± 0.01%) samples (P < .05). The researchers conclude that vitrification generates greater sperm alterations than freeze-drying and freezing, whereas freeze-drying produces lower LPO and higher ΔΨM for equine spermatozoa.

Relationship of cysteine-rich secretory protein-3 gene and protein with semen quality in stallions.

Cysteine-rich secretory protein-3 (CRISP-3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP-3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP-3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP-3 was identified and haplotypes were derived. Semen quality was assessed through the total motility (MOT), sperm vitality (SV), normal morphology (NM), functional integrity of membrane (MI) and a seminal quality index (SQi). CRISP-3 protein content of seminal plasma (SP) was determined by ELISA. The effect of the genotype, the haplotype and the concentration of the CRISP-3 protein on the seminal quality were evaluated through generalized linear models and linear regression analyses. Homozygous genotypes for SNP1, SNP2 and SNP3 and the heterozygous genotype for SNP4 showed a positive effect on seminal quality. Different haplotypes with positive effect on MOT, SV, NM, MI and SQi were identified. The allelic substitution analysis resulted in positive regression coefficients for MOT (SNP2) and MI (SNP2 and SNP3). A high level of CRISP-3 resulted in a higher MOT and SQi. It is concluded that the quality of stallion semen is influenced by the genotype of CRISP-3 and the concentration of CRISP-3 protein in SP.

Association of the cysteine-rich secretory protein-3 (CRISP-3) and some of its polymorphisms with the quality of cryopreserved stallion semen.

Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3) and CRISP3c.+716A>G (SNP4). CRISP-3 protein content in seminal plasma was determined by enzyme-linked immunosorbent assay. Semen from 30 stallions was cryopreserved and post-thaw motility, kinetics, abnormal morphology (AM), sperm vitality (SV) and membrane integrity (MI) were evaluated. Generalized linear models were fitted and means were compared using Tukey's test. Correlation and regression analyses were performed. For SNP1 and SNP3, the AA genotype had the highest results for motility and MI; for SNP2, the best results for motility and AM were obtained with the CC genotype. For SNP4, the GG genotype had the lowest results, except for MI. A high level of CRISP-3 protein in seminal plasma had the best results for motility, kinetics, SV and AM. In conclusion, there was a relationship between CRISP-3 genotype and seminal plasma protein and post-thawing semen quality in stallions.

Índices de selección y niveles independientes de descarte para dos características productivas y reproductivas en un hato holstein (Bos taurus) / Índice de seleção e níveis independentes de abate para dois características produtivas e reprodutivas em um rebanho holstein (Bos taurus) / Selection indexes and independent culling levels for two productive and reproductive traits in a holstein (Bos taurus) dairy herd

Con la finalidad de evaluar la selección mediante índices y niveles independientes de descarte (NID), para las variables producción de leche ajustada a 305 días (PL) y periodo abierto (PA), fueron analizados 1708 y 1206 registros para PL y DA, respectivamente. Mediante el programa MTDFREML se estimaron parámetros de heredabilidad de 0.23 ± 0.065 y 0.09 ± 0.074, para PL y PA, respectivamente; y repetibilidad de 0.34 ± 0.061 y 0.18 ± 0.054, para PL y PA, respectivamente. Las correlaciones genética, ambiental, y fenotípica se estimaron entre PL y PA, con valores de 0.15 ± 0.32, 0.42 y 0.34, respectivamente. Para la elaboración de Índices y NID, y para la estimación de sus progresos genéticos, fueron analizadas diferentes relaciones entre valores económicos relativos. Los mayores progresos fueron encontrados para unos valores económicos relativos de 1 a 3 (PA:PL). Así mismo, diferentes intensidades de selección fueron evaluadas encontrando para ambos casos que los mayores progresos genéticos fueron obtenidos para la máxima intensidad de selección utilizada (fracción seleccionada del 10%). Ambos métodos de selección demostraron la obtención de progresos genéticos simultáneos para los rasgos en estudio; sin embargo, de acuerdo con la estimación de una relación comparativa entre los progresos genéticos, se encontró que mediante el método de índices se proyectó un 12.6% más progreso genético por generación respecto del progreso estimado para NID. A pesar de esto y de ser el índice un método superior para la elección de los mejores animales, este requiere una estimación precisa de las correlaciones entre las características, lo cual no fue del todo factible en el desarrollo de este trabajo, por lo que se sugiere que el uso del NID es más conveniente para un programa de selección de esta naturaleza.

With the purpose to evaluate genetic selection by using indexes and independent culling levels (ICL) for productive and reproductive traits, 305-d milk yield (MY) and days open (DO), 1707 and 1206 records for MY and DO, respectively, were analyzed. By means of the MTDFREML program genetic parameters for heritability of 0.23 ± 0.065 and 0.09 ± 0.074 for MY and DO, respectively, and for repeatability of 0.34 ± 0.061 and 0.18 ± 0.054 for MY and DO, respectively, were estimated. Also the genetic, phenotypic and environmental correlations between MY and DO were estimated, resulting in values of 0.15 ± 0.32, 0.42 and 0.34, respectively. For the indexes and ICL elaboration, and the estimation of genetic progress, different relations between relative economic values were analyzed. The highest progress was found for DO:MY economic values at 1:3 ratio. In the same way, evaluations for different selection intensities were done, where the highest genetic progress was obtained by the used of the maximum selection intensity (selected fraction of 10%) for both cases. Although with both selection methods the achievement of simultaneous genetic progress for each trait was demonstrated, by estimating a comparative relationship between both genetic progresses, a 12.6% more genetic progress by each generation was found for Index than for ICL methods. Nevertheless, despite being the Index a better method for selection of the best animals it requires a more precise estimation of correlations between traits, a process that was not feasible performed in the present study. Thereafter, ICL is proposed as the more convenient for that type of selection program.

A fim de avaliar a seleção através de índices e níveis independentes de abate (NID), para as variáveis produção de leite ajustada para 305 dias (PL) e dia aberto (DA), foram analisados os registros para 1708 e 1206 MP e DA, respectivamente. Através do programa MTDFREML parâmetros da herdabilidade de 0,23 ± 0,09 ± 0.065 e 0.074, foram estimados para PL e PA, respectivamente, e parâmetros de repetibilidade de 0.34 ± 0.061 e 0.18 ± 0.054, para PL e PA, respectivamente. Correlações genéticas, fenotípicas e ambientais foram estimados entre PL e PA, com valores de 0.15 ± 0.32, 0.42 e 0.34, respectivamente. Para o desenvolvimento de indicadores e NID, e para estimar seu progresso genético relações econômicas entre os diferentes valores foram analisados. O maior avanço foi encontrado por algum parente valores econômicos de 1 a 3 (DA:PL). Do mesmo modo, diferentes intensidades de seleção foram avaliadas para ambos os casos, constataram que os maiores progressos genéticos foram obtido para a intensidade máxima de seleção utilizada (fração selecionados a partir de 10%). Ambos os métodos de seleção mostrou obtenção de progressos simultâneos características genéticas no estudo, mas de acordo com a estimativa de uma relação entre o progresso genético comparativo, verificou-se que utilizando o índice é projetado um aumento 12.6% progresso genético uma geração de progresso em matéria estimado para NID. Apesar disto, e se o índice for superior a um método para a escolha das melhores animais, isto requer uma estimativa precisa das correlações entre as características, o que não era totalmente viável para o desenvolvimento deste trabalho, de modo a uso de NID é sugerido é mais adequado para um programa de seleção desta natureza...