Effects of Obesity and Diabetes on Sperm Cell Proteomics in Rats.

Infertility caused by male factors is potentially associated with metabolic disorders such as obesity and/or diabetes. This experimental study was conducted in a male rodent model to assess the effects of different diseases on semen quality and sperm proteomics. Ten Wistar rats were used for each treatment. Rats were fed commercial food provided controllably to the control group and the diabetic group, and a hypercaloric diet supplemented with 5% sucrose in water was provided ad libitum to the obese group for 38 weeks. Diabetes was induced with 35 mg/kg streptozotocin. After euthanasia, testicles, spermatozoa, fat, and blood (serum) samples were collected. Spermatozoa were evaluated for quality and subjected to proteomics analyses. Histology and cytology of the testis, and serum leptin, adiponectin, interleukin 8 (IL-8), blood glucose, and testosterone levels, were also assessed. Body weight, retroperitoneal and testicular fat, and the Lee index were also measured. Obesity and diabetes were induced. The diabetic group showed noticeable changes in spermatogenesis and sperm quality. The mass spectrometry proteomics data have been deposited in Mendeley Data (doi: 10.17632/rfp7kfjcsd.5). Fifteen proteins varied in abundance between groups, especially proteins related to energy production and structural function of the spermatozoa, suggesting disturbances in energy production with a subsequent alteration in sperm motility in both groups, but with a compensatory response in the obese group.

Effect of refrigeration at -1°C on spermatozoa quality of domestic cats / Efeito da refrigeração a -1°C na qualidade de espermatozoides de gatos domésticos

The objective of this study was to evaluate the sperm quality obtained of domestic cats by electroejaculation and recovery of the tail of the epididymis after cooling at -1°C and 4°C for 24 and 48 hours. Twenty-nine adult cats (2 to 6kg) were used. Sperm collection was performed by electroejaculation (EEJ), and after 48 hours, the cats were orchiectomized, and sperm sample was obtained from the vas deferens and epididymis tail (EPD). The samples were diluted in ACP-117® extender, and the sperm characteristics were evaluated at three different moments: when still fresh, 24 and 48 hours after cooling. The objective of this study was to evaluate the sperm quality obtained of domestic cats by electroejaculation and recovery of the tail of the epididymis after cooling at -1°C and 4°C for 24 and 48 hours. Twenty-nine adult cats (2 to 6kg) were used. Sperm collection was performed by electroejaculation (EEJ), and after 48 hours, the cats were orchiectomized, and sperm sample was obtained from the vas deferens and epididymis tail (EPD). The samples were diluted in ACP-117® extender, and the sperm characteristics were evaluated at three different moments: when still fresh, 24 and 48 hours after cooling. In order to compare the two refrigeration temperatures, the first stage was to analyze if there was a difference between the harvesting techniques. After this, two experiments were conducted: in the first, sperm sample from 14 cats were used and the cooling was performed at -1°C; and in the second, sample from 15 cats were used and the sperm were refrigerated at 4°C. Sperm kinetics were evaluated by computerized analysis (CASA) and concentration by Neubauer chamber, spermatic morphology was evaluated by modified Karras staining, and membrane integrity was evaluated by eosin nigrosine. The results obtained were analyzed in R software, version 3.2.5 using the Mann-Whitney test for variables with abnormal distributions, considering significance at the level of 5%. In ejaculate samples, higher values of total morphological defects were observed after 24 and 48 hours of refrigeration at 4°C (P<0.022) compared to refrigeration at -1°C, using Friedman test. To quantify the decrease in sperm quality, parameter reductions were calculated among time points (F-24h/F-48h/24h-48h). In EPD samples, a greater reduction in sperm quality was detected after 24 hours of refrigeration at 4°C, both in motility and sperm kinetics and in the movement and velocity indices, compared to refrigeration at -1°C. Based on the results, it can be concluded that cooling of feline spermatozoa at -1°C for up to 48 hours was efficient in maintaining spermatic quality collected by EEJ and EPD, and it could be an alternative to spermatozoa cryopreservation in domestic felines.(AU)

O objetivo deste trabalho foi avaliar a qualidade espermática de gatos domésticos obtidos por eletroejaculação e recuperação da cauda do epidídimo após a refrigeração a -1°C e a 4°C por 24 e 48 horas. Vinte e nove gatos adultos (2 a 6kg) foram utilizados. A colheita de espermatozoides foi realizada por eletroejaculação (EEJ) e, após 48 horas, os gatos foram orquiectomizados, e as amostras espermáticas foram obtidas a partir do ducto deferente e da cauda do epidídimo (EPD). As amostras foram diluídas em ACP-117® e as características espermáticas foram avaliadas em três momentos distintos: fresco, 24 e 48 horas após a refrigeração. Para ser possível comparar as duas temperaturas de refrigeração, a primeira etapa foi analisar se havia diferença entre as técnicas de colheita. Após isto, dois experimentos foram conduzidos: no primeiro, espermatozoides de 14 gatos foram utilizados e a refrigeração foi realizada a -1°C; e no segundo, amostras de 15 gatos foram utilizados e os espermatozoides foram refrigerados a 4°C. A cinética espermática foi avaliada por análise computadorizada (CASA), a concentração por câmara de Neubauer, a morfologia espermática foi avaliada pela coloração de Karras modificada, e a integridade da membrana foi avaliada por eosina nigrosina. Os resultados obtidos foram analisados no software R, versão 3.2.5, utilizando o teste de Mann-Whitney para variáveis com distribuições anormais, considerando significância ao nível de 5%. No ejaculado, maiores valores de defeitos morfológicos totais foram observados após 24 e 48 horas de refrigeração a 4°C (P<0,022) em comparação com refrigeração a -1°C, usando o teste de Friedman. Para quantificar a diminuição na qualidade espermática, as reduções dos parâmetros foram calculadas entre os pontos de tempo (F-24h/F-48h/24h-48h). Na EPD, uma maior redução na qualidade espermática foi detectada após 24 horas de refrigeração a 4°C, tanto na motilidade e na cinética espermática quanto nos índices de movimento e velocidade, em comparação com a refrigeração a -1°C. Com base nos resultados, pode concluir-se que a refrigeração dos espermatozoides felino a -1°C, até 48 horas, foi eficaz na manutenção da qualidade espermático colhidos por EEJ e EPD, e pode ser uma alternativa para a criopreservação de espermatozoides em felinos domésticos.(AU)

Proteomic analysis of amniotic and allantoic fluid from buffaloes during foetal development.

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800 g for 10 min and then at 10,000 g for 60 min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50 µg) was used for in-solution tryptic digestion, and mass spectrometry analysis (nano-LC-MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty-one proteins were found in the amniotic fluid, and twenty-one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α > 1). Fibronectin and alpha-1-antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha-2-macroglobulin and alpha-2-HS-glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha-2-macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes.

Calcium chloride combined with dimethyl sulphoxide for the chemical sterilization of dogs.

The aim of this study was to evaluate the effect of intratesticular injection of calcium chloride (CaCl2 ) combined with dimethyl sulphoxide (DMSO) as a chemical sterilization in dogs. Twelve dogs were divided into two groups: the treated group (n = 6), in which 15 mg/kg of a 7.5% CaCl2 solution combined with 0.5% DMSO was injected into each testicle (volume range 1.0-4.76 ml); and a control group (n = 6) that received the same volume/kg of 0.9% sodium chloride solution (NaCl). Semen characteristics pre- and post-treatment were evaluated. Serum testosterone concentration was determined before the injection (D-1) and at 15 (D15), 30 (D30) and 60 (D60) days after intratesticular injection. Testicle sizes and local pain were evaluated for 7 consecutive days (D1 to D7) and at D15, D30 and D60 after injection. At D60, testicle histological evaluation was performed after orchiectomy. No pain was observed by testicular palpation, with the exception of one dog in the treated group; this dog then received analgesic therapy. An increase in testicular volume was evident within 24 hr after treatment, followed by gradual reduction for 3 weeks. Five of 6 dogs from the treated group presented azoospermia at D15; the remaining dog presented at D30. There was no significant difference in testosterone concentrations in the treated group during the experimental period. Histological evaluation showed testicular degenerative lesions, especially at the proximal and middle portions. The results indicated that one injection of 7.5% CaCl2 combined with 0.5% DMSO into each testis is a viable alternative for canine sterilization.