Chlorothalonil as a potential endocrine disruptor in male zebrafish (Danio rerio): Impact on the hypothalamus-pituitary-gonad axis and sperm quality.

Chlorothalonil is a broad-spectrum organochlorine fungicide widely employed in agriculture to control fungal foliar diseases. This fungicide enters aquatic environments through the leaching process, leading to toxicity in non-target organisms. Organic contaminants can impact organism reproduction as they have the potential to interact with the neuroendocrine system. Although there are reports of toxic effects of chlorothalonil, information regarding its impact on reproduction is limited. The aim of the present study was to evaluate the influence of chlorothalonil on male reproductive physiology using the zebrafish (Danio rerio) as ecotoxicological model. Zebrafish were exposed for 7 days to two concentrations of chlorothalonil (0.1 and 10 µg/L) along with a control group (with DMSO - 0.001%). Gene expression of hypothalamus-pituitary-gonad axis components (gnrh2, gnrh3, lhr, fshr, star, hsd17b1, hsd17b3, and cyp19a1), as well as hepatic vitellogenin concentration were assessed. In sperm cells, reactive oxygen species (ROS) content, lipid peroxidation (LPO), mitochondrial functionality, and membrane integrity and fluidity were evaluated. Results indicate that exposure to the higher concentration of chlorothalonil led to a reduction in brain gnr2 expression. In gonads, mRNA levels of lhr, star, and hsd17b1 were decreased at both chlorothalonil concentrations tested. Similarly, hepatic vitellogenin concentration was reduced. Regarding sperm cells, a decreased ROS level was observed, without significant difference in LPO level. Additionally, a higher mitochondrial potential and lower membrane fluidity were observed in zebrafish exposed to chlorothalonil. These findings demonstrate that chlorothalonil acts as an endocrine disruptor, influencing reproductive control mechanisms, as evidenced by changes in expression of genes HPG axis, as well as hepatic vitellogenin concentration. Furthermore, our findings reveal that exposure to this contaminant may compromise the reproductive success of the species, as it affected sperm quality parameters.

Sperm cell capacitation status of ram semen after cooling

Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifications similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5o C. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artificial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cooling, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extenders (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extenders provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time.

Fertility disorders in male dogs

Male dog fertility disorders are usually troublesome and challenging for a practicing veterinarian. It may be generally assumed, that reproductive potential in this species is lower than in farm animals and it is still decreasing. This situation starts to be similar to human medicine, where we observe dramatic drop of reproductive capacity, which resulted in the need of implementation of Assisted Reproductive Technologies (ART). Situation in dogs is more complicated owing the fact, that the use of ART meets many obstacles. Low fertility potential in dogs appears to be the result of variable factors such as: specific criteria of selection for reproduction in which fertility performance in not a priority, lack of periodical obligatory fertility check, species specific predisposition for many reproductive diseases and no age limit for reproductive use of males. Dogs are kept in human environment and exposition for civilizational byproducts influences negatively not only on our health, but also on health our 'minor brothers'. It should be bear in mind, that reproductive organs are very sensitive for environmental factors disrupting homeostatic balance. The decline in male dog fertility over the past decades was proved, with potential link to environmental contaminants (4). They were found in pet foods and were also detected in the sperm and testes of adult dogs causing a detrimental effects on sperm function. Over the 26 years of the study of Lea et al. (4), authors found a decrease in the percentage of normal motile sperm. Between 1988 and 1998, sperm motility declined by 2.5 per cent per year. Then from 2002 to 2014 sperm motility continued to decline at a rate of 1.2% per year. In addition, the male pups had an increased incidence of cryptorchidism. Basics of physiology of reproduction of male dogs. Normally the puberty in males is associated with presence of normal sperm cells in genital organs. It is reached in male dogs at age around 5-6 months. Such a young dog obviously cannot be used for reproduction. Reproductive maturity is associated later, with development of normal sexual behavior and production of sufficient number of normal, fertilizing competent spermatozoa. It corresponds with 12-18 months of animal age. Testicular descent is completed usually before weaning period, but sometimes testicles may reach scrotum later, but never after the end of 6 month of age. That time inguinal canals start to be so narrow, that caudal passing of gonads is unlikely. Male dogs have only one accessory sexual gland - prostate, which produces vast portion of seminal plasma.(AU)

Exogenous DNA length and quantity affect the transfection rate, but not sperm viability during sperm-mediated gene transfer

Spermatozoa have a spontaneous ability to take up exogenous DNA in a process regulated by specific mechanisms. This ability has been used to carry exogenous DNA into oocytes during fertilization to produce transgenic animals; a process called sperm-mediated gene transfer (SMGT). However, it is still an inefficient method and little is known about the effect of exogenous DNA once associated with spermatozoa, on sperm characteristics. Therefore, the objective of the present work was to evaluate the effects of exogenous DNA length and its amount on DNA uptake by bovine spermatozoa as well as spermatozoa viability. For that, spermatozoa (5 × 106 cells/mL) were incubated for 1 h at 38.5 °C with different exogenous DNA lengths (2.2, 5.5, or 8.5 kb) at different concentrations (number of molecules or ng). The association of exogenous DNA with spermatozoa was quantified by PCR real-time and the spermatozoa viability was evaluated by flow cytometry. Here, we show that no matter the amount of exogenous DNA used, larger sequences are less efficiently (p ˂ 0.05) associated with bovine spermatozoa. Besides that, the length and amount of exogenous DNA do not compromise sperm viability. Taken together, the results support that the length of exogenous DNA is more important than the amount used to influence its association with sperm cells. Thus, the size and quantity of exogenous DNA can be optimized to increase SMGT protocols, without altering the sperm viability.

Freezing of Stallion Semen: In Vitro Evaluation of Motility and Acrosin Activity in Sperm Cells Cryopreserved Using Different Semen Extenders.

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes and further immersion in liquid nitrogen at -196°C for long-term storage. After thawing, semen samples were initially evaluated for sperm motility, both total and progressive, and acrosin activity. Moreover, semen was incubated at 37°C and further assessed at 60 and 120 minutes in a thermoresistance test (TRT) for sperm motility and acrosin activity. Immediately after thawing, both progressive and total motility, and acrosin activity were lower (p < 0.05) in thawed semen than in fresh semen. During the TRT, total sperm motility and acrosin activity after 60 minutes were lower (p < 0.05) than those obtained after thawing. Similarly, total sperm motility and acrosin activity were lower (p < 0.05) after 120 minutes than at 60 minutes of the TRT. The analysis of motility and acrosin activity allowed the conclusion that both extenders have a similar capacity to preserve the integrity of sperm cells subject to freezing and thawing.

Nitrosative stress in human spermatozoa causes cell death characterized by induction of mitochondrial permeability transition-driven necrosis.

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.

Ultrastructural description of the spermatogenesis and spermatozoa in Phaenocora unipunctata (Platyhelminthes, Neotyphloplanida).

Ultrastructural studies of spermiogenesis and sperm morphology have found many characters that are likely to provide clues to the phylogeny of the Platyhelminthes. However, the lack of information on many free-living groups has been a limiting factor. There is a single description of the spermatogenesis and spermatozoa in a Phaenocora species, namely P. anomalocoela, therefore a similar analysis was made in Phaenocora unipunctata to compare the intrageneric variation of sperm ultrastructure and spermatogenesis in the Neotyphloplanida. The comparison of the two Phaenocora species shows that several characters have the potential to be relevant to hypothesize phylogenetic relationships at different taxonomic levels. The presence of superficially incorporated axonemes outside the ring of cortical microtubules in the mature sperm cell, resulting from the fusion of the axonemes with the median cytoplasmic process during spermiogenesis, as well as the presence of a constant number of microtubules in the different regions of the spermatozoon, seem to constitute apomorphies of the genus Phaenocora. Furthermore, the presence of an axonemal spur, the compression of cortical microtubules by the rotation of the basal bodies during spermiogenesis, and the presence of a connection between the nucleus and the plasma membrane in the mature spermatozoon, support previous proposals that these characters are apomorphies of Dalytyphloplanida. The comparison of spermatogenesis and spermatozoa of P. unipunctata and P. anomalocoela demonstrates that studying intrageneric variation can give valuable insights into the significance of many characters proposed for phylogenetic studies of the Rhabdocoela.

Acción de oxígeno en especies reactivas en los espermatozoides / Action of reactive oxygen species on spermatozoa / Ação das espécies reativas de oxigênio nos espermatozoides

Para que el esperma puede desempeñar sus funciones y lograr el objetivo final, que es la fertilización, requieren energía que proviene de trifosfato de adenosina (ATP). Durante el metabolismo de la energía se almacena en forma de electrones de alta energía que se dirige a la cadena de transporte de electrones, compuesto de cinco proteínas supramolecular. En este proceso, se produce la formación de una, el anión intermedio radical semiquinona (Q- ) que es finalmente capaz de transferir este electrón desapareado a O2 y formar especies reactivas del oxígeno, la superóxido principal, peróxido de hidrógeno y el radical hidroxilo. Estas moléculas son procesos de activación altamente reactivos y espermatozoides que llevan a la degradación y la disminución de la calidad celular. Sin embargo, estas mismas moléculas que a menudo se consideran grandes villanos, también participan en los mecanismos que culminan en la fertilización y la formación de cigoto, adquiriendo así una gran importancia. Con el advenimiento de la criopreservación del semen y el estrés oxidativo asociado con el procesamiento, especies reactivas de oxígeno entraron en evidencia en la investigación, por lo que es necesario estudiar su acción y formas de reducir el estrés oxidativo.(AU)

So that spermatozoa can perform functions and achieve the goal that is fertilization, require energy from adenosine triphosphate (ATP). During the energy metabolism is stored in the form of high-energy electrons that are directed to the electron transport chain, comprised of five supramolecular protein. In this process, there is the formation of a semiquinone anion (Q¯) an intermediate radical that is eventually able to transfer this unpaired electron to O2 and form reactive oxygen species, as the main superoxide, hydrogen peroxide and hydroxyl radical. These molecules are highly reactive and sperm trigger processes that lead to degradation and decrease in cell quality. However these same molecules that are often considered great villains, also participate in mechanisms that culminate in fertilization and zygote formation, thus acquiring great importance. With the advent of semen cryopreservation and oxidative stress associated with the processing, reactive oxygen species entered into evidence in the research, making it necessary to study its action and ways to reduce oxidative stress.(AU)

Para que os espermatozoides possam executar suas funções e alcançarem o objetivo final, que é a fecundação, necessitam de energia que advém da adenosina trifosfato (ATP). Durante o metabolismo, a energia é armazenada na forma de elétrons de alta energia que são direcionados a cadeia de transporte de elétrons, composta de cinco proteínas supramoleculares. Neste processo, há a formação de um radical intermediário, o ânion semiquinona (Q) que eventualmente é capaz de transferir este elétron desemparelhado ao O2 e formar as espécies reativas de oxigênio, sendo as principais o superóxido, o peróxido de hidrogênio e o radical hidroxila. Essas moléculas são altamente reativas e nos espermatozoides desencadeiam processos que levam à degradação e decréscimo na qualidade celular. Porém, essas mesmas moléculas que muitas vezes são consideradas grandes vilãs, também participam de mecanismos que culminam na fertilização e formação do zigoto, adquirindo assim uma grande importância. Com o advento da criopreservação de sêmen e o estresse oxidativo associado ao processamento, as espécies reativas de oxigênio entraram em evidência nas pesquisas, tornando-se necessário o estudo de sua ação e maneiras de reduzir o estresse oxidativo.(AU)

Acción de oxígeno en especies reactivas en los espermatozoides / Action of reactive oxygen species on spermatozoa / Ação das espécies reativas de oxigênio nos espermatozoides

Para que el esperma puede desempeñar sus funciones y lograr el objetivo final, que es la fertilización, requieren energía que proviene de trifosfato de adenosina (ATP). Durante el metabolismo de la energía se almacena en forma de electrones de alta energía que se dirige a la cadena de transporte de electrones, compuesto de cinco proteínas supramolecular. En este proceso, se produce la formación de una, el anión intermedio radical semiquinona (Q- ) que es finalmente capaz de transferir este electrón desapareado a O2 y formar especies reactivas del oxígeno, la superóxido principal, peróxido de hidrógeno y el radical hidroxilo. Estas moléculas son procesos de activación altamente reactivos y espermatozoides que llevan a la degradación y la disminución de la calidad celular. Sin embargo, estas mismas moléculas que a menudo se consideran grandes villanos, también participan en los mecanismos que culminan en la fertilización y la formación de cigoto, adquiriendo así una gran importancia. Con el advenimiento de la criopreservación del semen y el estrés oxidativo asociado con el procesamiento, especies reactivas de oxígeno entraron en evidencia en la investigación, por lo que es necesario estudiar su acción y formas de reducir el estrés oxidativo.

So that spermatozoa can perform functions and achieve the goal that is fertilization, require energy from adenosine triphosphate (ATP). During the energy metabolism is stored in the form of high-energy electrons that are directed to the electron transport chain, comprised of five supramolecular protein. In this process, there is the formation of a semiquinone anion (Q¯) an intermediate radical that is eventually able to transfer this unpaired electron to O2 and form reactive oxygen species, as the main superoxide, hydrogen peroxide and hydroxyl radical. These molecules are highly reactive and sperm trigger processes that lead to degradation and decrease in cell quality. However these same molecules that are often considered great villains, also participate in mechanisms that culminate in fertilization and zygote formation, thus acquiring great importance. With the advent of semen cryopreservation and oxidative stress associated with the processing, reactive oxygen species entered into evidence in the research, making it necessary to study its action and ways to reduce oxidative stress.

Para que os espermatozoides possam executar suas funções e alcançarem o objetivo final, que é a fecundação, necessitam de energia que advém da adenosina trifosfato (ATP). Durante o metabolismo, a energia é armazenada na forma de elétrons de alta energia que são direcionados a cadeia de transporte de elétrons, composta de cinco proteínas supramoleculares. Neste processo, há a formação de um radical intermediário, o ânion semiquinona (Q) que eventualmente é capaz de transferir este elétron desemparelhado ao O2 e formar as espécies reativas de oxigênio, sendo as principais o superóxido, o peróxido de hidrogênio e o radical hidroxila. Essas moléculas são altamente reativas e nos espermatozoides desencadeiam processos que levam à degradação e decréscimo na qualidade celular. Porém, essas mesmas moléculas que muitas vezes são consideradas grandes vilãs, também participam de mecanismos que culminam na fertilização e formação do zigoto, adquirindo assim uma grande importância. Com o advento da criopreservação de sêmen e o estresse oxidativo associado ao processamento, as espécies reativas de oxigênio entraram em evidência nas pesquisas, tornando-se necessário o estudo de sua ação e maneiras de reduzir o estresse oxidativo.

Viability of cryopreserved bull spermatozoa collected from the epididymis stored for 12 hours at 4ºC / Viabilidade dos espermatozoides criopreservados de touros, colhidos do epidídimo após 12 horas a 4ºC

The purpose of this study was to evaluate effect of epididymis cooling on bovine frozen sperm viability. Ten pairs of testes/epididymes were collected of a commercial slaughterhouse; one epididymis from each pair was refrigerated at 4ºC for 12 hours and the other immediately proceeded. Epididymis was isolated, sperm cells collected after epididymal slicing and then analyzed regarding to motility, vigor, total number of sperm, morphology and membrane integrity. Sperm cells were frozen in Botubov® extender (Botupharma Biotecnologia Animal, Botucatu, SP, Brazil) and thawed for semen analysis. The same procedure was performed with cooled testis/epididymis. Results demonstrated higher viability (p < = 0,05) of fresh cells to the total number of spermatozoa (1,9 ± 1,2 versus 0,9 ± 0,9 x 109 spermatozoa), sperm motility (74,0 ± 15,1 versus 20,5 ± 13,8%) and vigor (3,7 ± 0,5 versus 1,7 ± 0,8), and for pos-thawing motility (23,5 ± 16,7 versus 8,0 ± 7,9%) and vigor (2,0 ± 0,8 versus 0,8 ± 0,8) when spermatozoa were collected immediately post-slaughter than maintained cooling 12 hours, respectively. We conclude that 12 hours of epididymis cooling after slaughter decreases sperm cells quality.(AU)

O objetivo deste estudo foi avaliar o efeito da refrigeração do epidídimo, sobre a viabilidade dos espermatozoides congelados. Foram colhidos dez pares de testículos/epidídimos de um abatedouro comercial. Um dos epidídimos foi refrigerado a 4C durante 12 horas e o contralateral foi imediatamente processado. O epidídimo foi isolado, as células espermáticas extraídas e analisadas quanto à motilidade, vigor, concentração, morfologia e integridade da membrana. Após a análise, os espermatozoides foram congelados em diluente Botubov® (Botupharma Biotecnologia Animal, Botucatu, SP, Brasil) e descongelados para análise. O mesmo procedimento foi realizado com o testículo/epidídimo refrigerado. Os resultados evidenciaram maior viabilidade (p < = 0,05) das células pré-congelação para os parâmetros, número total de espermatozides (1,9 ± 1,2 versus 0,9 ± 0,9 x 109 espermatozoides), motilidade (74,0 ± 15,1 versus 20,5 ± 13,8%) e vigor (3,7 ± 0,5 versus 1,7 ± 0,8), e pós-congelação, motilidade (23,5 ± 16,7 versus 8,0 ± 7,9%) e vigor (2,0 ± 0,8 versus 0,8 ± 0,8) quando os espermatozoides foram colhidos a partir de epidídimos processados imediatamente após o abate quando comparados aos mantidos 12 horas sob refrigeração, respectivamente. Conclui-se que 12 horas de refrigeração do epidídimo após o abate, prejudica a qualidade das células espermáticas, impossibilitando a congelação do sêmen.(AU)

Viabilidade dos espermatozoides criopreservados de touros, colhidos do epidídimo após 12 horas a 4ºC / Viability of cryopreserved bull spermatozoa collected from the epididymis stored for 12 hours at 4ºC

O objetivo deste estudo foi avaliar o efeito da refrigeração do epidídimo, sobre a viabilidade dos espermatozoides congelados. Foram colhidos dez pares de testículos/epidídimos de um abatedouro comercial. Um dos epidídimos foi refrigerado a 4°C durante 12 horas e o contralateral foi imediatamente processado. O epidídimo foi isolado, as células espermáticas extraídas e analisadas quanto à motilidade, vigor, concentração, morfologia e integridade da membrana. Após a análise, os espermatozoides foram congelados em diluente Botubov® (Botupharma Biotecnologia Animal, Botucatu, SP, Brasil) e descongelados para análise. O mesmo procedimento foi realizado com o testículo/epidídimo refrigerado. Os resultados evidenciaram maior viabilidade (p≤0,05) das células pré-congelação para os parâmetros, número total de espermatozides (1,9 ± 1,2 versus 0,9 ± 0,9 x 109 espermatozoides), motilidade (74,0 ± 15,1 versus 20,5 ± 13,8%) e vigor (3,7 ± 0,5 versus 1,7 ± 0,8), e pós-congelação, motilidade (23,5 ± 16,7 versus 8,0 ± 7,9%) e vigor (2,0 ± 0,8 versus 0,8 ± 0,8) quando os espermatozoides foram colhidos a partir de epidídimos processados imediatamente após o abate quando comparados aos mantidos 12 horas sob refrigeração, respectivamente. Conclui-se que 12 horas de refrigeração do epidídimo após o abate, prejudica a qualidade das células espermáticas, impossibilitando a congelação do sêmen

The purpose of this study was to evaluate effect of epididymis cooling on bovine frozen sperm viability. Ten pairs of testes/epididymes were collected of a commercial slaughterhouse; one epididymis from each pair was refrigerated at 4ºC for 12 hours and the other immediately proceeded. Epididymis was isolated, sperm cells collected after epididymal slicing and then analyzed regarding to motility, vigor, total number of sperm, morphology and membrane integrity. Sperm cells were frozen in Botubov® extender (Botupharma Biotecnologia Animal, Botucatu, SP, Brazil) and thawed for semen analysis. The same procedure was performed with cooled testis/epididymis. Results demonstrated higher viability (p≤0,05) of fresh cells to the total number of spermatozoa (1,9 ± 1,2 versus 0,9 ± 0,9 x 109 spermatozoa), sperm motility (74,0 ± 15,1 versus 20,5 ± 13,8%) and vigor (3,7 ± 0,5 versus 1,7 ± 0,8), and for pos-thawing motility (23,5 ± 16,7 versus 8,0 ± 7,9%) and vigor (2,0 ± 0,8 versus 0,8 ± 0,8) when spermatozoa were collected immediately post-slaughter than maintained cooling 12 hours, respectively. We conclude that 12 hours of epididymis cooling after slaughter decreases sperm cells quality.

Utilização de pentoxifilina e antioxidantes na criopreservação do sêmen bovino: qualidade seminal e estresse oxidativo / Utilization of pentoxifylline and antioxidants in cryopreservation of bovine semen: semen quality and oxidative stress

O presente estudo avaliou a adição da pentoxifilina, tocoferol, ascorbato e suas combinações sobre a proteção da célula espermática bovina contra os efeitos deletérios da criopreservação. Foram utilizados 24 touros Nelores (Bos taurus indicus), criados em sistema semi-intensivo. Foi coletado um ejaculado de cada reprodutor, diluídos em TRIS-citratogema-glicerol e divididos em seis partes. Cada parte foi suplementada da seguinte maneira: sem aditivos (controle), tocoferol (10 mmol/mL), tocoferol (10 mmol/mL) + pentoxifilina (1mg/mL), ascorbato (0,45mg/mL), ascorbato (0,45mg/mL) + pentoxifilina (1mg/mL) ou pentoxifilina (1mg/mL). Após o descongelamento, as amostras foram avaliadas quanto à motilidade e características do movimento, integridade da membrana plasmática e de acrossomo e atividade mitocondrial. Os níveis de peroxidação lipídica espontânea e induzida foram avaliados pela produção de substâncias reativas ao ácido tiobarbitúrico (TBARS). A suplementação com pentoxifilina, tocoferol, ascorbato e suas combinações, não alterou (P>0,05) a atividade mitocondrial, integridade acrossomal, e a concentração de TBARS espontâneo e induzido. A adição de tocoferol + pentoxifilina reduziu a motilidade progressiva quando comparado ao ascorbato e também a integridade da membrana espermática quando comparado ao controle e ao ascorbato (P˂0,05). Já a adição de ascorbato + pentoxifilina foi deletéria sobre linearidade em comparação ao tratamento ascorbato (P˂0,05). A adição de ascorbato, tocoferol e pentoxifilina individualmente ou em combinação, não foi eficiente em diminuir os danos causados pela criopreservação e estresse oxidativo em amostras pós descongelamento de sêmen bovino.(AU)

The present study evaluated the addition of pentoxifylline, tocopherol and ascorbate and their combinations on the protection of bovine spermatic cells against the deleterious effects of cryopreservation. A total of 24 Nelore bulls (Bos Taurus indicus) were included in this study, raised in a semi-intensive system. One ejaculation was collected from each bull and diluted in tris-citrate-yolk-glycerol, divided into six portions, and then supplemented with: no additives (control); tocopherol (10 mmol/ml), tocopherol (10 mmol/ml) + pentxoifylline (1 mg/ml), ascorbate (0.45 mg/ml), ascorbate (0.45 mg/ml) + pentoxifylline (1 mg/ml) and pentxoifylline (1 mg/ml). After thawing, the samples were evaluated for motility and characteristics of movement, acrosomal and plasma membrane integrity, and mitochondrial activity. Spontaneous and induced lipid peroxidation levels were evaluated by the production of thiobarbituric acid reactive substances (TBARS). Supplementation with pentoxifylline, tocopherol and ascorbate and their combinations, did not alter mitochondrial activity, acrosomal integrity, or the spontaneous and induced TBARS concentration (P>0.05). The ascorbate, tocopherol and pentoxifylline additions, both individually and in combinations, were inefficient in decreasing the damage caused by cryopreservation and oxidative stress in post-thawed bovine semen samples.(AU)

Utilização de pentoxifilina e antioxidantes na criopreservação do sêmen bovino: qualidade seminal e estresse oxidativo / Utilization of pentoxifylline and antioxidants in cryopreservation of bovine semen: semen quality and oxidative stress

O presente estudo avaliou a adição da pentoxifilina, tocoferol, ascorbato e suas combinações sobre a proteção da célula espermática bovina contra os efeitos deletérios da criopreservação. Foram utilizados 24 touros Nelores (Bos taurus indicus), criados em sistema semi-intensivo. Foi coletado um ejaculado de cada reprodutor, diluídos em TRIS-citratogema-glicerol e divididos em seis partes. Cada parte foi suplementada da seguinte maneira: sem aditivos (controle), tocoferol (10 mmol/mL), tocoferol (10 mmol/mL) + pentoxifilina (1mg/mL), ascorbato (0,45mg/mL), ascorbato (0,45mg/mL) + pentoxifilina (1mg/mL) ou pentoxifilina (1mg/mL). Após o descongelamento, as amostras foram avaliadas quanto à motilidade e características do movimento, integridade da membrana plasmática e de acrossomo e atividade mitocondrial. Os níveis de peroxidação lipídica espontânea e induzida foram avaliados pela produção de substâncias reativas ao ácido tiobarbitúrico (TBARS). A suplementação com pentoxifilina, tocoferol, ascorbato e suas combinações, não alterou (P>0,05) a atividade mitocondrial, integridade acrossomal, e a concentração de TBARS espontâneo e induzido. A adição de tocoferol + pentoxifilina reduziu a motilidade progressiva quando comparado ao ascorbato e também a integridade da membrana espermática quando comparado ao controle e ao ascorbato (P˂0,05). Já a adição de ascorbato + pentoxifilina foi deletéria sobre linearidade em comparação ao tratamento ascorbato (P˂0,05). A adição de ascorbato, tocoferol e pentoxifilina individualmente ou em combinação, não foi eficiente em diminuir os danos causados pela criopreservação e estresse oxidativo em amostras pós descongelamento de sêmen bovino.

The present study evaluated the addition of pentoxifylline, tocopherol and ascorbate and their combinations on the protection of bovine spermatic cells against the deleterious effects of cryopreservation. A total of 24 Nelore bulls (Bos Taurus indicus) were included in this study, raised in a semi-intensive system. One ejaculation was collected from each bull and diluted in tris-citrate-yolk-glycerol, divided into six portions, and then supplemented with: no additives (control); tocopherol (10 mmol/ml), tocopherol (10 mmol/ml) + pentxoifylline (1 mg/ml), ascorbate (0.45 mg/ml), ascorbate (0.45 mg/ml) + pentoxifylline (1 mg/ml) and pentxoifylline (1 mg/ml). After thawing, the samples were evaluated for motility and characteristics of movement, acrosomal and plasma membrane integrity, and mitochondrial activity. Spontaneous and induced lipid peroxidation levels were evaluated by the production of thiobarbituric acid reactive substances (TBARS). Supplementation with pentoxifylline, tocopherol and ascorbate and their combinations, did not alter mitochondrial activity, acrosomal integrity, or the spontaneous and induced TBARS concentration (P>0.05). The ascorbate, tocopherol and pentoxifylline additions, both individually and in combinations, were inefficient in decreasing the damage caused by cryopreservation and oxidative stress in post-thawed bovine semen samples.

Sperm concentration on the intrauterine artificial insemination in swine / Concentrações espermáticas na inseminação artificial intra-uterina suína

The aim of this work was to evaluate the efficiency of the intrauterine insemination (IUI) in swine, considering the conception rate, farrowing rate, litter size (alive born pigs). For the IUI, the females had been insemination at 24 and 48 hours after the estrus detection, and the inseminating doses of 500 million, 1 billion, 1.5 billion and 2 billion spermatozoa in 20 mL extender had been used. The procedure of catheter insertion through the cervical canal was successfully performed in 97.9 percent of the females. The conception rate was 6.3 percent in the IUI. The farrowing rate in IUI was 87.2 percent but the farrowing rate was 100 percent for the sperm concentration of 500 million. Regarding the number of born pigs and alive born pigs observed in females inseminated with IUI, no significant difference was observed (p > 0.05). The concentration of 500 x 10(6) spermatozoa in 20 mL extender in the intrauterine insemination resulted in an optimal reproductive performance.

Conduziu-se este estudo, com o objetivo de avaliar a eficiência da inseminação intra-uterina (IIU) em suínos, considerando as taxas de retorno ao estro, aborto, parto, além do tamanho da leitegada (número de leitões nascidos e nascidos vivos). Na IIU, as fêmeas foram inseminadas nos tempos de 24 e 48 horas após a detecção do estro, utilizando-se as concentrações de 500 milhões, 1 bilhão, 1,5 bilhão e 2 bilhões de espermatozóides, em 20mL de diluente. A passagem do cateter de IIU através da cérvix foi possível em 97,9 por cento das fêmeas. Foi realizado diagnóstico de retorno ao estro a partir do 18º dia e diagnóstico de gestação por ultrassonografia transcutânea entre o 28º e 30º dias após a inseminação. A taxa de retorno ao estro foi de 6,3 por cento na IIU. A taxa de parto na IIU foi de 87,2 por cento, sendo a taxa de parto para a concentração de 500 milhões de 100 por cento. Com relação ao número de leitões nascidos totais e nascidos vivos, não houve diferenças, entre as diferentes concentrações espermáticas (P>0,05). A utilização da concentração de 500 x 10(6) espermatozóides em 20mL de diluente, com inseminação intra-uterina, obteve-se um bom desempenho reprodutivo.