Sperm characteristics, peroxidation lipid and antioxidant enzyme activity changes in milt of Brycon orbignyanus cryopreserved with melatonin in different freezing curves.

The objective of this study was to evaluate extender supplemented with melatonin and freezing curves on the antioxidant enzyme activity, peroxidation lipid and sperm characteristics of cryopreserved Brycon orbignyanus milt. Males (n = 16) and females (n = 5) were hormonally induced with two doses (0.5 mg and 5.0 mg kg-1) of carp pituitary extract, and their gametes were collected by light abdominal massage. The fresh milt was diluted at a ratio of 1:4 (milt:extender) in the following solutions: (Control) 10% methyl glycol (MG) + 5% Beltsville thawing solution; (M1) Control + 1 mM melatonin; and (M2) Control + 2 mM melatonin. The freezing curves were C1 (automated freezer) and C2 (dry shipper for 24 h). After each curve was recorded, the straws were transferred to a liquid nitrogen container until the analyses were performed. The samples were thawed in a water bath (60 °C, 8 s) and evaluated using the Sperm Class Analyzer software for the parameters total motility, progressive motility, curvilinear velocity, straight line velocity, mean displacement velocity, straightness, and linearity. The following were also measured: motility time, vitality, morphology, oxidative stress (lipid peroxidation, activity of catalase and superoxide dismutase, quantification of nitric oxide), and fertilization and hatching rates. The data were analyzed within R by one-way analysis of variance and Tukey's test for comparison of means (p < 0.05). A significant difference (p < 0.05) was observed between the solutions in vitality, morphology, motility, and fertilization rate, the solutions with melatonin having the best values. Total motility, progressive motility, and motility time were significantly different. Among oxidative stress markers, only lipid peroxidation and superoxide dismutase activity showed an effect of the curve × solution interaction (p < 0.05), the solutions with melatonin yielding the lowest values. The fertilization and hatching rates were also higher under the melatonin treatments, regardless of the curve. Melatonin 2 mM and slow curve are indicated for the cryopreservation of fish species sperm as it led to the slowest detrimental spermatozoa effects and better fertilization and hatching rates.

Impact of activation solutions on fresh and frozen-thawed sperm motility and fertilization success for two species of migratory freshwater fishes.

Extracellular environment conditions, ionic concentration, pH, osmolality, and temperature influence sperm activation and sperm quality. The aim of this study was to evaluate the effects of different activating solutions on sperm quality and fertilization rate of fresh and post-thaw sperm in Brycon orbignyanus and Prochilodus vimboides. Activation solutions with ions: NaCl, KCl, CaCl2 (150 mOsm kg-1), without ions: glucose (150 mOsm kg-1) and water: reverse osmosis (RO) (∼0 mOsm kg-1) and tank water (TW) were tested. In experiment 1, fresh sperm motility was activated in each activating solution and motility rate (%), motility quality score (0-5), and motility duration (seconds) were subjectively evaluated using a microscope. In experiment 2, sperm was cryopreserved and post-thaw sperm quality was assessed in each activating solution. Methyl glycol was used as cryoprotectant and for B. orbignyanus a solution of BTS® 5% in water reverse osmosis - 325 mOsm kg-1 was used as extender, while for P. vimboides it was used a solution of glucose 5% in water reverse osmosis - 325 mOsm kg-1. In straw, cryoprotectant, extender, sperm were respectively 10%, 80% 10% (V/V). B. orbignyanus fresh sperm activated in NaCl, KCl, glucose solutions, TW and RO yielded higher averages for all the subjective parameters analysed. In fresh sperm of P. vimboides the highest values were observed when glucose solution was used for sperm motility activation, and the highest fertilization rates were observed in samples activated in glucose or RO solutions. B. orbignyanus post-thaw sperm activated in TW (45.1%) or RO (39.7%) presented the highest values for motility. The highest values of curvilinear velocity (VCL) were observed using glucose (69.5 µm s-1), NaCl (67 µm s-1) and KCl (68.4 µm s-1), but the highest fertilization rates were observed when glucose (3.6%), RO (3.5%) and TW (2.5%) were used. P. vimboides post-thaw sperm activated in glucose solution presented the highest motility rate (41%), VCL (43.7 µm s-1), fertilization rate (18.2%) and hatching rate (13.7%). In order to achieve the best seminal quality, fertilization and hatching rates in both fresh and post-thaw sperm, the glucose solution, TW and RO are indicated for use as sperm motility activators in B. orbignyanus, whereas for P. vimboides the glucose solution and RO are indicated.

In vitro fertilization of porcine oocytes is affected by spermatic coincubation time / A fertilização in vitro de ovócitos suínos é afetada pelo tempo de coincubação espermático

The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF), and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte). The sperm and oocytes were coincubated according to the following treatments (T): T1 = oocytes exposed to spermatozoa for one hour (173 oocytes), T2 = oocytes exposed to spermatozoa for two hours (170 oocytes), and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes). After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium) to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm). Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P<0.05) after two hours of coincubation time than it was for one or three hours. Furthermore, 68.60% of the ova coincubated with the spermatozoa for two hours were monospermic. The oocytes exposed to spermatozoa for one hour (T1) presented a higher (P<0.01) rate of polyspermy than those in T2 and T3. Fertilization performance (%) did not differ (P>0.05) between oocytes exposed to spermatozoa for one (T1) and three hours (T3). However, optimum (P=0.048) results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P>0.05) between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.(AU)

Esse estudo foi realizado para avaliar os efeitos de diferentes tempos de coincubação dos gametas sobre a fertilização in vitro (FIV) de suínos e se a eficiência dessa técnica poderia ser melhorada pela redução no período que os ovócitos são expostos aos espermatozoides durante a FIV. Um total de 508 (em grupos de 50) complexos cumulus-ovócito (COCs) imaturos foram maturados no meio NCSU-37. Os COCs foram cultivados por 40-44 horas e então inseminados com sêmen in natura (2.000 espermatozoides/ovócito). Os espermatozoides e ovócitos foram coincubados de acordo com os seguintes tratamentos (T): T1 = ovócitos expostos aos espermatozoides por uma hora (173 ovócitos); T2 = ovócitos expostos aos espermatozoides por duas horas (170 ovócitos) e T3 = ovócitos expostos aos espermatozoides por três horas (165 ovócitos). Após esses períodos de coincubação, os ovócitos foram lavados em meio de fertilização (meio TALP) para remoção dos espermazoides não ligados a zona pelúcida e cultivados em outro mesmo meio (não contendo espermatozoides). Após 18-20 horas de fertilização, os prováveis zigotos foram corados com Hoechst-33342 para avaliação dos resultados da FIV. A taxa de penetração foi maior (P<0,05) após o tempo de coincubação de duas horas em comparação a uma e três horas. Além disso, 68,60% dos ovócitos coincubados com os espermatozoides por duas horas foram monospérmicos. Os ovócitos expostos aos espermatozoides por uma hora (T1) apresentaram elevada (P<0,01) taxa de polispermia em comparação com o T2 e T3. A eficiência da fertilização (%) não diferiu (P>0,05) entre os ovócitos expostos aos espermatozoides por uma (T1) e três horas (T3). Entretanto, ótimos (P=0,048) resultados foram obtidos após duas horas de coincubação, quando a taxa da eficiência da fertilização foi 50,16 ± 8,52%. O número de espermatozoides penetrados por ovócito e a formação de pro-núcleo masculino não diferiu (P>0,05) entre os tratamentos avaliados. Sob as condições de ensaio realizadas, especialmente em relação à concentração espermática utilizada, a coincubação dos gametas por um período de duas horas parece ser ótima para as taxas de monospermia e eficiência da fertilização. Portanto, um tempo provavelmente ótimo para obter um elevado número de embriões suínos capazes de ter um desenvolvimento normal.(AU)

Toxicity of cryoprotectants on Prochilodus lineatus (Valenciennes, 1837) (curimba) embryos in an experimental incubator (Characiformes: Prochilodontidae)

This paper investigated the effect of cryoprotectant substances on Prochilodus lineatus embryos in an experimental incubator. The prospective study applied combinations of polyvinyl alcohol, hydroxyethyl cellulose, gelatin and fetal bovine serum with dimethyl sulfoxide and ethylene glycol in a new experimental incubator. The morphology of embryos, larval viability and the efficiency of experimental incubators in maintaining the quality of embryos were evaluated. This study demonstrates the efficient association between hydroxyethylcellulose and dimethyl sulfoxide as greater viability (p<0.05) was found for embryos (72.9 ± 23.9%). It should also be noted the permeation of cryoprotectants in embryos through the changes found in chorion diameter, embryo diameter and embryo volume comparing the treatments versus control group (water) (p<0.05), this results can help in future cryopreservation protocols. Although the temperature and oxygenation differed between the usual and experimental incubators (p<0.05), the results showed a high fertilization rate (79.6 ± 13.2%) for experimental incubators (p<0.05) which is sufficient for the maintenance of embryos in a cryoprotective environment and effectively allows experimentation for long periods with cryoprotectant substances. Cryopreservation of fish embryos has not been accomplished yet and new approaches are required for understanding the permeability of teleost embryos, especially in Brazilian native species.

Este trabalho avaliou o efeito de substâncias crioprotetoras sobre embriões de Prochilodus lineatus em uma incubadora experimental. O estudo aplicou combinações de álcool polivinílico, hidroxietilcelulose, gelatina e soro fetal bovino com dimetilosulfóxido e etilenoglicol em uma nova incubadora experimental. Foram avaliadas a morfologia dos embriões, a viabilidade larval e a eficiência das incubadoras experimentais na manutenção da qualidade dos embriões. Este estudo demonstra a associação eficiente entre hidroxietilcelulose e dimetilsulfóxido pela maior viabilidade (p<0,05) encontrada para os embriões (72,9 ± 23,9%). Deve-se notar também a permeação dos crioprotetores nos embriões através das alterações encontradas no diâmetro córion, diâmetro do embrião e no volume do embrião comparando os tratamentos ao grupo controle (água) (p<0,05), estes resultados podem ajudar em futuros protocolos de criopreservação. Embora a temperatura e a oxigenação diferiram entre as incubadoras comuns e as experimentais (p<0,05), os resultados mostraram elevada taxa de fertilização (79,6 ± 13,2%) para incubadoras experimentais (p<0,05), o que é suficiente para a manutenção de embriões em ambiente crioprotetor e permite efetivamente a experimentação por longos períodos com substâncias crioprotetoras. A criopreservação de embriões de peixes ainda não foi realizada e novas abordagens são necessárias para a compreensão da permeabilidade dos embriões de teleósteos, especialmente em espécies nativas brasileiras.

Porcine follicular fluid concentration of free insulin-like growth factor-I collected from different diameter ovarian follicles / Concentração do fator de crescimento semelhante à insulina tipo I no fluido folicular suíno coletado a partir de folículos ovarianos de diferentes diâmetros

The study aimed to quantify the concentrations of free IGF-I in serum and fluid of ovarian follicles in pre-pubertal gilts and describe the ovarian morphology by measuring the size of the ovaries and counting the number of surface follicles. Ovaries (n=1,000) from pre-pubertal gilts were obtained immediately after slaughter. A total of 10 samplings were performed, with ovaries obtained from 50 females for each collection. The follicles situated on the surface of each ovary were classified as small (SFs, 2 to 5mm in diameter) or large (LFs 6 to 10mm in diameter) and the follicular fluid was obtained by follicle aspiration. The collection of serum samples was performed after the gilts exsanguination using sterile tubes. From the pool of serum and follicular fluid obtained from 50 females, the concentration of free IGF-I was determined in each sample using an enzyme immunoassay kit (ELISA). The description of ovarian morphometry was performed in 100 ovaries from randomly selected gilts. The larger and smaller lengths of ovaries were measured, and the total number of SFs and LFs present on the surface of each ovary were also counted. The IGF-I concentration was greater (P<0.05) in LFs (170.92±88.29 ng/mL) compared with SFs (67.39±49.90ng/mL) and serum (73.48±34.63ng/mL). The largest and smallest length of the ovaries was 26.0±3.0 and 19.0mm ±2.0mm, respectively. The number of SFs (70.86±25.76) was greater (P<0.01) than LFs (6.54±5.26). The study concluded that LFs present greater levels of IGF-I when compared with SFs and blood, which is related to increased activity of the LFs and its differentiation to ovulation. In addition, ovaries of pre-pubertal gilts have a higher number of SFs compared to LFs. Therefore, our study demonstrated unique data regarding the physiological concentration of free IGF-I in ovarian follicles, that can be used in future research to evaluate the addition of this hormone in the in vitro production media of porcine embryos with the...

Objetivou-se quantificar as concentrações do IGF-I livre no soro e no fluido de folículos ovarianos de fêmeas suínas pré-púberes e descrever a morfologia ovariana, por meio da mensuração das dimensões dos ovários e da contagem do número de folículos superficiais. Ovários (n=1.000) foram obtidos de fêmeas pré-púberes imediatamente após o abate. Foi realizado um total de 10 coletas, sendo em cada, obtidos ovários de 50 fêmeas. Os folículos localizados na superfície de cada ovário foram classificados em pequenos (FPs, 2-5mm de diâmetro) ou grandes (FGs, 6-10mm de diâmetro) e o fluido folicular foi obtido por aspiração dos folículos. A coleta do soro foi realizada após a exsanguinação das fêmeas com o uso de tubos estéreis. A partir do pool de fluido folicular e do soro obtido das 50 fêmeas, determinou-se a concentração de IGF-I livre em cada amostra por meio de kit de ensaio imunoenzimático (ELISA). A descrição da morfometria ovariana foi realizada em 100 ovários provenientes de fêmeas escolhidas aleatoriamente. Foi mensurado o comprimento maior e menor dos ovários e, também, contabilizado o número total de FPs e FGs presentes na superfície de cada ovário. A concentração de IGF-I foi superior (P<0,05) nos FGs (170,92±88,29ng/mL) em comparação com os FPs (67,39±49,90ng/mL) e o sérico (73,48±34,63ng/mL). O comprimento maior e menor dos ovários foi de 26,0±3,0mm e 19,0±2,0 mm, respectivamente. O número de FPs (70,86±25,76) foi maior (P<0,01) em comparação com os FGs (6,54±5,26). Conclui-se que FGs apresentam níveis de IGF-I superiores aos FPs, e ao sangue, sendo isso relacionado a maior atividade dos FGs e à diferenciação que os mesmos sofrem para a ovulação. Além disso, ovários de fêmeas suínas pré-púberes apresentam elevado número de FPs em comparação aos FGs. Portanto, nosso estudo demonstrou dados originais a respeito da concentração fisiológica de IGF-I livre em folículos ovarianos, que podem ser utilizados em futuras pesquisas para avaliar a adição de...

Vitamin E and reduced glutathione in Prochilodus lineatus (curimba) semen cryopreservation (Characiformes: Prochilodontidae)

This study investigated the addition of antioxidants vitamin E and reduced glutathione on curimba (Prochilodus lineatus) semen cryopreservation and compared sodium bicarbonate solution and distilled water as activators. The experiment was conducted at the environmental station of CEMIG, in Itutinga-MG, Brazil, between December/2009 and January/2010. Semen samples (n = 7) with semen motility above 80% were diluted in cryoprotectant solutions composed of 10% methanol, 15% lactose and containing different concentrations of antioxidants: 50 (VE50), 100 (VE100) and 250 (VE250) µM of vitamin E, and 0.5 (RG0.5), 1.0 (RG1.0) and 1.5 (RG1.5) mM of reduced glutathione. A solution without antioxidants was used as a control. The semen was diluted at a ratio of 1:4 (100 ìL semen:400 ∝L cryoprotectant solution). The toxicity of the solutions was evaluated by investigating semen motility after 10 min in the solution. The rest of the diluted semen was placed into 0.5 mL straws maintained in nitrogen vapour for 24 hours and packed into a nitrogen liquid cylinder for four days. The samples were thawed in a water bath at 60°C for 8 s and the rate (%) and duration (s) of semen activation with distilled water or sodium bicarbonate was evaluated. In the toxicity test, we found that vitamin E and reduced glutathione were not toxic to curimba semen at any of the tested concentrations (P>0.05). The duration of motility was longer (P<0.05) in semen activated with sodium bicarbonate 1% (163 ± 11 s), which was considered the best activator for semen under these conditions. No significant differences were found between the cryoprotectant solutions used after freezing (P>0.05). Thus, the antioxidants vitamin E and reduced glutathione did not improve the quality of cryopreserved curimba semen, but they did not cause toxic effects to the semen in natura and they did not decrease its quality during cryopreservation.

Este estudo avaliou a adição de antioxidantes vitamina E e glutationa reduzida no sêmen criopreservado de curimba (Prochilodus lineatus) e comparou solução de bicarbonato de sódio e água destilada como ativadores. O experimento foi conduzido na estação ambiental da CEMIG, em Itutinga-MG, entre Dezembro/2009 e Janeiro/2010. Sêmen de sete animais, com motilidade espermática acima de 80%, foi diluído em soluções crioprotetoras compostas por metanol 10% e lactose 15% em diferentes concentrações de antioxidantes: 50 (VE50), 100 (VE100) e 250 (VE250) µM de vitamina E, 0,5 (RG5.5), 1,0 (RG1.0) e 1,5 (RG1.5) mM glutationa reduzida e uma solução controle sem antioxidante. O sêmen foi diluído na proporção de 1:4 (100 µL de sêmen: 400 µL de solução crioprotetora). A toxicidade das soluções foi avaliada pela motilidade espermática após de 10 minutos em solução. O restante do sêmen diluído foi armazenado em palhetas de 0,5 mL mantidos em vapor de nitrogênio por 24 horas e estocado em cilindro de nitrogênio líquido por quatro dias. As amostras foram descongeladas em banho-maria a 60°C por 8 segundos e avaliada a taxa (%) e duração (s) pela ativação do sêmen com água destilada e bicarbonato de sódio a 1%. No teste de toxicidade, observamos que os antioxidantes da vitamina E e glutationa, nas diferentes concentrações, não foram tóxicos para o sêmen do curimba (P>0,05). A duração da motilidade foi maior (P<0,05) no sêmen ativado com bicarbonato de sódio, sendo o melhor ativador para o sêmen nestas condições. Não houve diferenças significativas entre as soluções crioprotetoras utilizadas para sêmen antes e após congelamento (P>0,05). Assim, os antioxidantes vitamina E e glutationa reduzida não melhoram a qualidade do sêmen criopreservado de curimba, mas não causam efeitos tóxicos para o sêmen in natura e criopreservados por não diminuir sua qualidade durante a criopreservação.