Eneiva Carla Carvalho Celeghini

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical‐chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT – cooling rate of −0.55 °C min−1 and freezing rate of −19.1 °C min−1) and automated (AT – cooling rate of −0.23 °C min−1 and freezing rate of −15 °C min−1), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein‐conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fisher’s test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 ± 1.41% and 30.50 ± 1.06%, with ethylene glycol was 21.17 ± 1.66% and 21.67 ± 1.13% and with dimethyl formamide was 8.33 ± 0.65% and 9.17 ± 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 ± 1.49% (CT) and 15.83 ± 1.26% (AT) to glycerol, 9.20 ± 1.31% (CT) and 9.92 ± 1.29% (AT) to ethylene glycol 4.65 ± 0.93% (CT) and 5.17 ± 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation

The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma membrane disorders (mean ± SEM 9.48 ± 0.65 and 1.62 ± 0.23% in raw and frozen-thawed sperm, respectively). However, both membrane peroxidation and protein phosphorylation were increased (P < 0.05) compared to raw semen (1.74 and 5.20-fold, respectively). There was a correlation (r = 0.73; P < 0.05) between the increase in lipid peroxidation and tyrosine phosphorylation. Seminal plasma, regardless of origin, reduced (P > 0.05) tyrosine phosphorylation present on the surface of cryopreserved sperm; however, lipid peroxidation was not significantly reduced. In conclusion, we inferred that emergence of phosphorylated proteins on the surface of cryopreserved sperm was due to increased lipid peroxidation that occurred during the freezing/thawing process; however, reduced tyrosine phosphorylation that occurred after addition of seminal plasma was triggered by other mechanisms, apparently independent from the reduction in membrane peroxidation.

Assessment of in vitro sperm characteristics and their importance in the prediction of conception rate in a bovine timed-AI program

The aims of this study were to assess in vivo fertility and in vitro sperm characteristics of different sires and to identify sperm variables important for the prediction of conception rate. Multiparous Nelore cows (n = 191) from a commercial farm underwent the same timed artificial insemination (timed-AI) protocol. Three batches of frozen semen from three Angus bulls were used (n = 9). A routine semen thawing protocol was performed in the laboratory to mimic field conditions. The following in vitro sperm analyses were performed: Computer Assisted Semen Analysis (CASA), Thermal Resistance Test (TRT), Hyposmotic Swelling Test (HOST), assessment of plasma and acrosomal membrane integrity, assessment of sperm plasma membrane stability and of lipid peroxidation by flow cytometry and assessment of sperm morphometry and chromatin structure by Toluidine Blue staining. For statistical analyses, Partial Least Squares (PLS) regression was used to explore the importance of various sperm variables in the prediction of conception rate. The following in vitro sperm variables were determined to be important predictors of conception rate: total motility (TM), progressive motility (PM), TM after 2 h of thermal incubation (TM_2 h), PM after 2 h of thermal incubation (PM_2 h), Beat Cross Frequency after 2 h of thermal incubation (BCF_2 h), percentage of rapidly moving cells after 2 h of thermal incubation (RAP_2 h), intact plasma membrane evaluated by HOST, intact plasma and acrosomal membranes evaluated by flow cytometry, intact plasma membrane suffering lipid peroxidation, major defects, total defects, morphometric width/length ratio, Fourier_0 and Fourier_2 and Chromatin Heterogeneity. We concluded that PLS regression is a suitable statistical method to identify in vitro sperm characteristics that have an important relationship with in vivo bull fertility.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen‐thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer‐assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate‐conjugated Pisum sativum agglutinin and 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post‐centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen‐thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Avaliação das características seminais de galos selecionados para a reprodução pelo desenvolvimento da crista

A selecao de galos para a reproducao pelo desenvolvimento da crista vem sendo empregada rotineiramente na avicultura, sendo necessarios estudos para verificar a eficiencia deste metodo de selecao. Este trabalho teve por objetivo comparar as caracteristicas seminais de galos selecionados para a reproducao pelo desenvolvimento da crista. Foram utilizados 65 galos matrizes da linhagem AgRoss, selecionados na 20a semana de idade e divididos em dois grupos: grupo A, animais sem crista desenvolvida (n=33), e grupo B, animais com crista desenvolvida (n=32). Foram realizadas colheitas semanais de semen dos galos de 24 a 71 semanas de idade, para avaliar as caracteristicas seminais. Foram realizados os testes t de Student e de Wilcoxon para comparar os grupos e analises de perfis para verificar efeitos do grupo, idade e interacao grupo x idade. Os valores de volume seminal, concentracao espermatica, motilidade e vigor foram maiores (p < 0,01) para o grupo B no periodo de 24-31 semanas de idade. A percentagem de defeitos espermaticos para o grupo A foi maior nos periodos de 24-27 (p < 0,01) e de 32-35 semanas de idade (p < 0,05). O peso corporal foi maior (p < 0,05) para os animais do grupo B no periodo entre 24-39 semanas de idade e para os galos do grupo A no periodo de 60-71 semanas de idade. Foram observados efeitos da idade e interacao grupo x idade para as caracteristicas seminais, exceto para vigor. Em conclusao, a observacao do desenvolvimento da crista na 20a semana de idade e um metodo eficiente na selecao de galos para a reproducao.

Existem relações entre tamanho e morfoecogenicidade do corpo lúteo detectados pelo ultra-som e os teores de progesterona plasmática em receptoras de embriões eqüinos?

O corpo luteo (CL) e a glândula produtora de progesterona (P4), hormonio cuja secrecao continua e essencial para o inicio e a manutencao da gestacao em femeas equinas, e, consequentemente, para a aplicabilidade de inumeras biotecnicas de reproducao. Considerando-se a importância do CL para a manutencao de uma gestacao normal e suas caracteristicas anatomofisiologicas, objetivou-se determinar por ultra-sonografia (US) o tamanho e a morfoecogenicidade (ME) do CL em receptoras de embrioes equinos desde a ovulacao (D0) ate nove dias apos (D9), bem como os niveis plasmaticos de P4 produzida no mesmo periodo. Para tanto, 57 eguas receptoras de um programa de transferencia de embrioes foram examinadas diariamente por US transretal desde a primeira deteccao dos sinais de estro ate o D9. A cada exame, os CL foram mensurados e sua ME registrada segundo escore de 1 a 6 (1=anecoico; 6=hiperecoico). Amostras de sangue foram colhidas diariamente e a P4 dosada por radioimunoensaio. O diagnostico de gestacao foi realizado por US aos 13 e 25 dias apos a ovulacao. Houve uma tendencia de os corpos luteos apresentarem ME crescente (de 1 a 5) desde o dia da ovulacao ate o D9. Os niveis de P4 foram < 2,16 ng/ml ate o D3, com consequente elevacao e manutencao em niveis de diestro entre D4 e D9 (3,41 a 4,33 ng/ml). O tamanho luteinico nao diferiu, com excecao das medias extremas durante o periodo (D2 = 31,54 mm versus D8 = 25,95mm; p < 0,05). Assim, o aumento da ME media dos CLs avaliados por US e acompanhado por aumento na concentracao plasmatica de P4 em receptoras de embrioes, mas este evento parece nao ser dependente do tamanho da glândula luteinica. Nao existe diferenca na ME, no tamanho dos corpos luteos, nem nos niveis de P4 circulante do D0 ao D9 em receptoras de embrioes equinos que se tornaram gestantes ou nao apos a transferencia de embrioes.

An Efficient Technique to Detect Sperm Reactive Oxygen Species: The CellRox Deep Red ® Fluorescent Probe

Heat stress and testicular traumas increase sperm Reactive Oxygen Species (ROS) resulting in Oxidative Stress (OS) and injury of sperm. The fluorescent probe CellROX Deep Red® is not already used to detect ROS in spermatozoa. On this way, this study aims to evaluate its efficiency in detecting ROS in ram sperm. For that, it was used ejaculates from rams performing the in vitro induction of sperm OS in T0, T50 and T100. T0 was semen sample that was not submitted to OS induction, T50 was 50% induced to OS induction and T100 was entire submitted to OS induction. The production of ROS was detected by CellROX Deep Red®. Data polynomial regression analysis was performed and the result of determination coefficient was 0.728. Besides, sixteen rams were submitted to scrotal insulation during 72 hours performing the in vivo induction of OS sperm. Analyses of sperm motility, morphology, DNA fragmentation and ROS (detected by CellROX Deep Red®) were done two times before the scrotal insulation and two times afterwards. Data analysis of variance was performed from the periods (before x after) and it is observed that after scrotal insulation the total and progressive motility decrease (p<0.05) and total and major sperm defects and sperm DNA fragmentation increase (p<0.05). Moreover, after insulation, it was observed increase (p<0.05) in sperm showing moderate and intense OS. Thus, it is possible to conclude that CellROX® fluorescent probe is able to detect ROS production in ram sperm by in vitro and in vivo induction being a new technique to detect sperm OS.

Superovulação de novilhas da raça Nelore com diferentes doses de FSH/LH e congelação de embriões pelo método one-step com etilenoglicol

The objetive of this study was to identify the better dose between 300 (n = 20), 400 (n = 21) and 500 IU (n = 21) of FSH/LH to stimulate Nelore heifers. The superovulation treatment started on day 10 (D0 = estrous) of the estrous cycle in 8 decreasing aplications for 4 days. The embryo recovery was achieved on day 6.5 after the first artificial insemination. The superovulatory response for 300, 400 and 500 IU FSH/LH was follicles (15.12, 15.76 and 14.94); corpus luteum (10.68, 11.55 and 10.81) and transferable embryos (5.20, 1.81 and 2.76). The 300 IU of FSH/LH group presented the best results in regard to transferable embryos. The transferable embryos were cryopreserved by one-step method with 1.5 M of ethylene-glycol, resulting in 8 pregnancies (7.5%) of 106 embryos transferred by non-curgical method. The 300 IU of FSH/LH presented better superovulatory response in comparison with 400 and 500 IU in Nelore heifers. The transfer of Bos taurus indicus embryos cryopreserved by one-step method in 1.5 M of ethilene glycol was not efficient.

Effect of cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on the integrity and functionality of cryopreserved bovine spermatozoa.

Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100, and 150 μM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 μM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation.

Addition of Antioxidants Myoinositol, Ferulic Acid, and Melatonin and Their Effects on Sperm Motility, Membrane Integrity, and Reactive Oxygen Species Production in Cooled Equine Semen

Abstract The present study aimed to evaluate the influence of myoinositol (MYO), ferulic acid (FA), and melatonin (MEL) in equine cooled semen. Ejaculates were collected and distributed into the following four treatments: MYO, FA, MEL, and control. A skim milk–based extender was used. Samples were cooled at 5°C and evaluated at 0, 4, and 8 hours after storage for motility, plasma and acrosomal membranes integrity, mitochondrial potential, and production of reactive oxygen species (ROS). Motility characteristics were not affected by treatment, except for the amplitude of lateral head displacement, which was higher in MYO (8.3 ± 0.2) compared with the control group (7.8 ± 0.2). No difference was observed among treatments for intact plasma membrane (%). However, the percentage of cells with intact plasma and acrosomal membranes and high mitochondrial potential was greater in the MEL (78.1 ± 2.0) and FA groups (78.8 ± 1.7) compared with the control group (73.8 ± 2.0). The high mitochondrial potential (%) was also greater in groups treated with MEL (80.1 ± 1.9) and FA (81.0 ± 1.5) compared with the control group (76.6 ± 2.0). In addition, percentage of cells with intact acrosome membrane was greater in MEL group (99.7 ± 0.1) compared with all other treatments. ROS production was not affected by treatments. In conclusion, FA and MEL provided the best protection to mitochondria, acrosome, and plasma membranes, suggesting that the addition of these antioxidants to equine semen extender can improve sperm quality. HighlightsFerulic acid and melatonin provided the best protection to all membranes.Melatonin increases the percentage of cells with intact acrosome.No difference was observed among treatments for intact plasma membrane (%).Motility characteristics were not affected, except for the amplitude of lateral head displacement (higher in myoinositol).Reactive oxygen species production was not affected by treatments.