Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%).In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

Background Maternal periodontal disease leads to low birth weight (LBW), insulin resistance (IR), increased TNF‐α levels, and alterations in insulin signaling in adult offspring. TNF‐α has been associated with the stimulation of IKKβ/NF‐κB, resulting in the decreased expression of GLUT4. Another mechanism that may be involved in decreasing GLUT4 expression is DNA methylation. This study aimed to evaluate in the adult offspring of rats with periodontal disease: IR, inflammatory pathways, DNA methylation, and expression of GLUT4. Methods Female Wistar rats were distributed into control and experimental periodontal disease groups. Seven days after induction of periodontal disease, both groups were mated with healthy male rats. After weaning, male offspring were distributed into control offspring (CN‐o) and periodontal disease offspring (PED‐o) groups. Body weights were measured from 0–75 days of age. At day 75, the following were measured in the offspring: IR (HOMA‐IR index); TNF‐α and NF‐κBp65 content in the gastrocnemius muscle (GM) by western blotting; IKKα/β, JNK, ERK 1/2, NF‐κBp65, and NF‐κBp50 phosphorylation status in the GM by western blotting; DNA methylation by restriction digest and real‐time PCR(qAMP); and expression of GLUT4 mRNA in the GM by real‐time PCR. Results LBW, IR, increases in TNF‐α, IKKα/β, ERK 1/2, NF‐κBp65, and NF‐κBp50 decreased expression of GLUT4 mRNA were observed in the PED‐o rats. No differences were identified in JNK phosphorylation status and DNA methylation in the evaluated regions of the GLUT4‐encoding gene Slc2a4. Conclusion Maternal periodontal disease causes LBW, IR, activation of inflammatory pathways, and decreased GLUT4 expression in the GM of adult offspring.

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < or = 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < or = 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

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This study aimed to characterise canine flow cytometry semen analysis, as well as seminal reactive oxygen species dosage using the Golden Retriever breed as model of study. Moreover, we searched for the influence of muscular dystrophy in Golden Retriever dogs on semen parameters. Thirty-seven semen samples were obtained from healthy Golden Retrievers (n = 15) and from muscular dystrophy affected dogs (n = 22). Sperm-rich fractions were analysed by standardised breeding soundness examination in addition to the assay of fluorescence assisted cell sorting for acrosome integrity, mitochondrial activity and DNA fragmentation. Volume of ejaculate, per cent of motile spermatozoa and vigour were similar between groups; there were no differences in the per cent of minor and major defects. Integrity of acrosomal membrane, mitochondrial potential and sperm DNA fragmentation had no significant differences between groups either. Animals from control group had higher concentration of spontaneous seminal oxidative species in comparison with affected animals. Dogs affected by dystrophy had seminal parameters similar to those observed in healthy dogs except for the lower concentration of oxidative species. Future studies aiming to establish reference values for canine seminal parameters should be considered preferably with distinction of breeds.

Hypertensive individuals taking anti‐hypertensive drugs from renin‐angiotensin system inhibitors may exhibit a more severe evolution of the disease when contracting the SARS‐CoV‐2 virus (COVID‐19 disease) due to potential increases in ACE2 expression. The study investigated ACE1 and ACE2 axes and hydroxychloroquine in the lungs and adipose tissue of male and female normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs). SHRs were treated with losartan (10 mg/kg/day) or captopril (10 mg/kg/day) for 14 days or 7 days with hydroxychloroquine (200 mg/kg/day) in drinking water. WKY rats were also treated for 7 days with hydroxychloroquine. Blood pressure (BP), protein, and mRNA expression of ACE1 and ACE2 were analyzed in serum, adipose, and lung tissues. Losartan and captopril reduced BP in both sexes in SHR, whereas hydroxychloroquine increased BP in WKY rats. Losartan reduced ACE2 in serum and lungs in both sexes and in adipose tissue of male SHRs. Captopril decreased ACE2 protein in the lung of females and in adipose tissue in both sexes of SHRs. Hydroxychloroquine decreased ACE1 and ACE2 proteins in the lungs in both sexes and adipose tissue in male SHRs. In female WKY rats, ACE2 protein was lower only in the lungs and adipose tissue. Losartan effectively inhibited ACE2 in male and captopril in female SHRs. Hydroxychloroquine inhibited ACE2 in male SHRs and female WKY rats. These results further our understanding of the ACE2 mechanism in patients under renin‐angiotensin anti‐hypertensive therapy and in many trials using hydroxychloroquine for COVID‐19 treatment and potential sex differences in response to drug treatment.

Sloths are animals that suffer with the destruction and fragmentation of forests. They experience a low population growth rate and need to be studied further for the preservation of the species. The objective of this study was to contribute data relevant to the reproductive physiology of this species, selecting a semen collection method and evaluating seminal characteristics that have never before been described in the literature. Fifteen Bradypus tridactylus males were captured in Manaus, Brazil. Nine of them were captured during the first half of 2004 (Group 1) and the others during the second half (Group 2). The animals were anesthetized with an i.m. injection of a combination of ketamine (10 mg/kg) and xylasine (1 mg/kg). Semen was collected by electroejaculaton using a rectal probe designed for domestic cats. Electrostimulations were given with a 0-100 mA/0-12 V variable electrostimulator in sequences of three progressive intensities, with ten repetitions at each intensity and variation of 10 mA between them. They started with 20 mA and peaked at 60 mA. Each stimulus lasted about 3 s. It was not possible to define the best intensity of stimulus to use and ejaculation could take place at any time of the stimulation (Fisher's exact test). Sperm motility and vigor were immediately analyzed. Sperm count was determined in a Neubauer chamber at a 1:50 (v:v) dilution in formol-saline. Morphology was examined at the same dilution. Fresh semen smears were made and stained using Spermac Stain� (Minit�b, Tiefenbach, Germany) protocol for a better evaluation of the spermatozoa acrosome and midpiece. In both methods 200 cells were counted for morphological evaluation. All animals ejaculated approximately 30 �L to 90 �L of semen. In some ejaculates the semen was too thin and flowed down the penis, so that the volume effectively collected was not sufficient for a complete spermiogram. Spermatozoa presented a wide variety of defects, and some physical characteristics differed (not significantly) between samples collected during the first and second halves of the year. Motility and vigor were very low, the sperm did not show forward progression, only oscillatory movement. However, a high percentage (80%) of spermatozoa were moving. The concentration in Group 1 ranged from 5000 spermatozoa/mm3 to 685 500 spermatozoa/mm3 (mean � 218 571.4 � 242 499.4). Sperm concentation was not assessed in Group 2. The morphology of the head could be elongated or squared, or the head could have a base narrower than the apex. The tail showed a unique feature: the midpiece narrowed abruptly, forming a nip in its transition to the tail. This was similar in appearance to the segmental aplasia of the mitochondrial sheath, but it was considered normal because it was observed in all spermatozoa. Although further studies are necessary to standardize the semen evaluation of sloths and to define the best protocol for electroejaculation, this pioneering study has shown the characteristics of sloth spermatozoa and the possibility of collecting semen throughout the electroejaculation process in this species. This work was supported by Fapesp 03/07457-4.