Influence of different warming temperatures on the vitrified testicular fragments from pre-pubertal cat.

Testicular vitrification is an alternative to preserve the genetic material of pre-pubertal animals. However, there are few studies on post-vitrification warming. Hence, the aim was to compare the influence of different warming temperatures on vitrified testicular fragments from pre-pubertal cats. The testicles were fragmented and divided into a control group (non-vitrified) and vitrified, using an association between dimethylsulphoxide and glycerol. The vitrified fragments were warmed at 50, 55 and 60°C/5 s. Morphological and morphometric evaluations were carried out using classical histology. Afterwards, the mitochondrial activity was evaluated using Rhodamine 123. The data were expressed in mean and standard error. The differences were considered significant when p < .05. In the histomorphological analysis, the testicular fragment presented seminiferous tubules with poorly developed germinal epithelium, compatible with pre-pubertal animals. The group warmed at 50°C presented similar to the control regarding the maintenance of the integrity of the tubules and cells, without stromal rupture and lamina propria alteration, as well as regarding the maintenance of the junctions between the cells. The group warmed at 55°C showed reduction of the cell junctions, and the one warmed at 60°C had increased detachment of the basement membrane (p < .05). The warming caused a reduction in the tubular diameter inversely proportional and progressive to the increase in temperature, with the highest diameter in the control group and the lowest in the 60°C group. The control group showed a lower incidence of Rhodamine 123, followed in ascending order of the warmings at 55 and 60°C. The higher mitochondrial activity was obtained with 50°C, showing an increase of the metabolic cell function at this temperature. It was concluded that the testicular fragment of pre-pubertal cats presents a better preserved morphology, morphometry and viability when warmed at 50°C.

Recovery and cryopreservation of epididymal sperm from domestic cat using powdered coconut water (ACP-117c) and TRIS extenders.

Cryopreservation of cats epididymal spermatozoa allows the conservation of the genetic material and the study of the cryogenic effect applied to the gametes of other felines. However, this biotechnique still presents variable results, being necessary the investigation of alternative extenders. Powdered coconut water (ACP-117c) has been efficient in the sperm freezing of several species and in the cat sperm refrigeration. Therefore, we aimed to evaluate the effect of the freezing stages and the quality of the cats' epididymal spermatozoa after thawing, using ACP-117c. Epididymides (n = 36) from 18 cats were processed using TRIS (n = 18) or ACP-117c (n = 18) for sperm recovery. The sperm were immediately evaluated. Then, this was cooled, glycerolized, frozen and thawed, and re-evaluated at each stage for sperm kinetics by Computer Assisted Semen Analysis, viability, functionality (HOST), mitochondrial activity (DAB) and morphology. There was a reduction in total motility and progressive motility after thawing in both groups, and TRIS was superior to ACP-117c. The curvilinear velocity reduced after thawing with ACP-117c. Viability decreased after glycerolization in TRIS. Although it also reduced after thawing in both groups, it was higher in TRIS. There was no change on HOST. Mitochondrial activity decreased during the cryopreservation steps for both extenders. Nevertheless, TRIS presented a higher percentage of spermatozoa from DAB class I and II after thawing. Morphology did not differ between extenders. Therefore, ACP-117c is an alternative for the recovery of cat epididymal spermatozoa; however, it is not efficient for freezing. Glycerolization and thawing are the most critical stages, regardless of the extender.

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca).

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ±â€¯7.7%) and membrane functionality (60.5 ±â€¯4.2%) and higher mitochondrial activity (21.5 ±â€¯3.7%) than those preserved in ACP-117c (20.9 ±â€¯5.4% motile sperm; 47.1 ±â€¯2.5% functional membrane; 11.8 ±â€¯1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ±â€¯3.3%) in comparison to samples diluted in ACP-117c (18.6 ±â€¯1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.

Extra Virgin Coconut Oil as a Cryoprotector for Cryopreservation of Caprine Semen.

The study aimed to evaluate the effect of powdered coconut water-based diluent (ACP-101c) associated with extra virgin coconut oil (CO) as an external cryoprotectant in the conservation of cryopreserved buck sperm. For cryopreservation, the ejaculates from four bucks were pooled and divided into three aliquots and diluted at 37°C for treatments T1 (ACP-101c + 2.5% egg yolk +7% glycerol), T2 (ACP-101c + 2.5% CO +7% glycerol), and T3 (ACP-101c + 5% CO +7% glycerol). Then, the samples were packaged and cooled at a rate of 1.07°C/min decrease. Upon reaching 4°C, the samples were stored in a refrigerator at 4°C for 30 minutes for stabilization. After this period, the straws were frozen in nitrogen vapor for 15 minutes and then immersed and stored in liquid nitrogen at -196°C. After thawing, the samples were evaluated for sperm kinetics, plasma membrane integrity, acrosomal integrity, membrane functionality, mitochondrial activity (MA), and sperm morphology. In this study, no statistically significant differences were observed between the three treatments regarding the kinetic parameters (p > 0.05; Table 1). However, in relation to the velocities, a reduction was observed beyond the expected. There were no statistically significant differences between the diluents T1, T2, and T3 for the three velocities (curvilinear velocity [VCL], linear velocity [VSL], average path velocity [VAP]). Furthermore, no statistically significant differences were observed (p > 0.05) among treatments regarding the evaluation of membrane integrity, the functional membrane, MA, and sperm morphology after thawing. In conclusion, the use of CO in concentrations of 2.5% and 5.0% is effective in maintaining goat sperm quality, presenting itself as an alternative diluent for international programs of artificial insemination and embryo transfers. [Table: see text].

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS.

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP-106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.

Characterization of seminal parameters, sperm morphometry, micromorphology, and ultrastructure in gray brocket deer (Mazama gouazoubira, Fischer, 1814).

Populations of gray brocket deer (Mazama gouazoubira) are declining; yet, knowledge on the reproductive biology of this species remains limited. Therefore, this study aimed to describe morphology, viability, membrane integrity, mitochondrial activity, morphometry, micromorphology, and ultrastructure of the gray brocket deer sperm. Three adult male gray brocket deer were used in the study. Semen collection was performed using electroejaculation. Semen were analyzed by evaluating pH, motilities, vigor, mass movement, volume, concentration, viability, membrane integrity, mitochondrial activity, morphology, and morphometry. Micromorphology and ultrastructure of sperm were analyzed using scanning and transmission electron microscopy (SEM and TEM), respectively. There was no significant difference among males regarding on pH, motilities, vigor, mass movement, volume, concentration, viability. High values for membrane integrity, mitochondrial activity, and normal sperm were observed. The most frequent defects were simple bent tail and bowed midpiece. The head length, and width, midpiece, and tail length were 8.5, 4.4, 11.5, and 41.3 µm, respectively. SEM sperm showed paddle-shaped heads, with apical ridge and serrated band on the equatorial segment. TEM revealed the nucleus, acrosome, plasma membrane, mitochondria sheath, proximal centrioles, segmented columns, axoneme, outer dense fibers, and fibrous sheath. SEM and TEM showed the presence of some abnormalities. These results are expected to provide baseline values of diverse semen parameters, contributing toward the development of reproductive biotechnologies for gray brocket deer and, other deer species at risk of extinction.

Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

Atuação do médico veterinário especialista em reprodução no manejo de canis / Performance of veterinary specialist in reproduction on kennels management

A criação de cães de raça vem aumentando significativamente, consequentemente a exigência de serviços veterinários no mercado para lidar com essa categoria específica também aumentou. Em especial, temos os serviços de reprodução que crescem devido a procura por melhoria da qualidade de plantel, uma vez que pela incrementação de biotécnicas é possível dinamizar o potencial de crescimento da criação. Logo, é ideal que o profissional que irá prestar este serviço seja altamente capacitado para sua realização, tendo noções não somente de reprodução, mas também de outras necessidades que a criação de cães requer. É importante que o profissional saiba selecionar os animais adequados a reprodução, tanto reprodutores, quanto matrizes, além de quando realizar os procedimentos, assim, evitar protocolos desnecessários e que possam comprometer a criação. Desta forma, o profissional pode fornecer o serviço mais completo possível, fazendo com que esse profissional seja referência no mercado.(AU)

The breeding of purebred dogs has increased significantly, consequently the demand for veterinary services on the market to deal with this specific category has also increased. In particular, we have reproduction services that grow due to the demand for improving the quality of the herd, since by increasing biotechniques it is possible to boost the growth potential of creation. Therefore, it is ideal that the professional who will provide this service is highly qualified for its performance, having notions not only of reproduction, but also of other needs that dog breeding requires. It is important that the professional knows how to select the appropriate animals for reproduction, both breeders and matrices, in addition to when to perform the procedures, thus avoiding unnecessary protocols that could compromise the creation. In this way, the professional can provide the most complete service possible, making this professional a reference on the market.(AU)

Reprodução de procionídeos / Procyonids reproduction

Poucas informações a respeito da biologia dos procionídeos estão disponíveis para quem busca conhecer mais sobre essas espécies. Entretanto, alguns estudos a respeito da reprodução destes animais foram realizados. As principais espécies encontradas no Brasil são o quati, o guaxinim e o jupará, e, dentre esses, o quati se destaca por ser o que possui o maior número de informações reprodutivas. A maioria dos procionídeos aparece como espécies não ameaçadas, porém a falta de informações sobre estes animais pode fazer com que estes dados possam estar defasados. É importante o conhecimento reprodutivo dos procionídeos pois, devido as ações antrópicas que ocasionam a perda de habitat natural, essas espécies, em breve, podem entrar para a lista de animais ameaçados de extinção. Assim, esse artigo tem como objetivo descrever alguns dados por meios de estudos reprodutivos que podem auxiliar ao desenvolvimento de estratégias de conservação dessas espécies.(AU)

Little information about the biology of procyonids is available for those who want to know more about these species. However, some studies regarding the reproduction of these animals were developed. The main species found in Brazil are coati, raccoon, and kinkajou, among which the coati stands out for being the one with the highest number of reproductive information. Most procyonids appear as non-threatened species, but the lack of information about these animals may cause these data to be outdated. Reproductive knowledge of procyonids is important because, due to anthropic actions that cause loss of natural habitat, these species may soon enter on the list of endangered animals. Thus, this article aims to describe some data by means of reproductive studies that can help to develop conservation strategies for these species.(AU)

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.(AU)

Effect of adding anethole to the cryopreservation medium (powdered coconut water) on morphology, kinetics, and oxidative stress of buck sperm / Efeito da adição de anethole ao meio de criopreserção (água de coco em pó) na morfologia, cinética e estresse oxidativo do sêmen de bode

The quality of post-thawing goat sperm is critical to the success of artificial insemination protocols and may be influenced by extenders, cryoprotectants, and antioxidant substances. Therefore, the objective of this study was to evaluate the effects of the antioxidant anethole on goat sperm diluted in preservation medium based on powdered coconut water (ACP-101c) and frozen. For that, each ejaculate was submitted to the following treatments: ACP-101c (control); control plus supplementation with 30, 300, or 2000 µg/ mL anethole. The samples were thawed and evaluated for morphology, kinetics, membrane integrity, and reactive oxygen species (ROS). The addition of anethole increased morphological abnormalities (P < 0.05), however, it did not affect sperm kinetics. Flow cytometry analysis showed that sperm cells cryopreserved with 300 µg/mL anethole had lower acrosome integrity than those cryopreserved in other treatments. Evaluation of oxidative stress revealed that cells stored in the presence of 2000 µg/mL anethole had small amounts of ROS when compared to those preserved in the control medium alone or supplemented with 300 µg/mL anethole (P < 0.05). After cryopreservation of sperm with 2000 µg/mL anethole, the highest percentage of viable sperm without ROS was observed (P < 0.05). In conclusion, despite reducing ROS levels, the supplementation of anethole in ACP-101c did not affect sperm kinetics or membrane integrity post-thawing, however, it did cause morphological damage to sperm.(AU)

A qualidade do espermatozoide caprino pós-descongelação é crítica para o sucesso dos protocolos de inseminação artificial e pode ser influenciada por extensores, crioprotetores e substâncias antioxidantes. Portanto, o objetivo deste estudo foi avaliar os efeitos do antioxidante anetole sobre espermatozoides caprinos diluídos em meio de conservação à base de água de coco em pó (ACP-101c) e congelados. Para tanto, cada ejaculado foi submetido aos seguintes tratamentos: ACP-101c (controle); controle mais suplementação com 30, 300 ou 2000 µg / mL de anetole. As amostras foram descongeladas e avaliadas quanto à morfologia, cinética, integridade de membranas e espécies reativas de oxigênio. A adição de anetole aumentou as anormalidades morfológicas (P < 0,05), no entanto, não afetou a cinética dos espermatozoides. A análise da citometria de fluxo mostrou que as células de esperma criopreservadas com 300 µg / mL anethole tinham integridade acrosma menor do que aquelas criopreservadas em outros tratamentos. A avaliação do estresse oxidativo revelou que as células armazenadas na presença de 2000 µg / mL anethole apresentaram pequenas quantidades de ROS quando comparadas às preservadas em meio de controle isoladamente ou suplementadas com 300 µg / mL anethole (P < 0,05). Após a criopreservação de espermatozoides com 2000 µg / mL anethole, observou-se a maior porcentagem de espermatozoides viáveis sem ROS (P < 0,05). A população com espermatozoides viáveis sem ROS foi maior quando utilizado 2.000 µg / mL (P < 0,05). Em conclusão, apesar de reduzir os níveis de ROS, a suplementação de anetole em ACP-101c não afetou a cinética espermática e a integridade da membrana pós-descongelação, entretanto, causou danos morfológicos nos espermatozoides.(AU)

Sêmen ovino e caprino imunossexado e diluído em meio de conservação à base de água de coco em pó (ACP101/102c) / Ram and goat semen immunosexed and diluted in powdered coconut water-based preservation medium (ACP101/102c)

Sperm sexing aims to separate sperm populations in carriers of the "X" or "Y" chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c. Ejaculates from five rams and five goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with "Y" chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P < 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species. The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.(AU)

Aspectos econômicos e avaliação do período de viabilidade de espermatozoides ovino imunossexados e diluídos em meio de conservação à base de água de coco em pó (ACP-102c) / Economic aspects and evaluation of the viability period of immunosexed and diluted ram sperm in powdered coconut water conservation extender (ACP-102c)

Background: Sperm sexing is increasing in use because pre-determining the sex of the calf allows greater profitability and promotes significant gains in the productive systems that utilize the technique. Deployment of a low-cost and practical preservation methodol-ogy may further favor the cost-benefit ratio. Flow cytometry, the most commonly used sexing technique, has high costs and is very restricted. As an alternative, immunosexing has been studied, which uses sex-specific monoclonal antibodies. Thus, the objective of this study was to evaluate the immunosexing technique in conjunction with cryopreservation in ACP-102c and examine its economic aspects with regard to ram semen.Materials, Methods & Results: Ejaculates from two ram individuals were collected with the aid of an artificial vagina, evaluated, and submitted to the immunosexing protocol, according to the manufacturer’s recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro-RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP (ACP-102c + 20% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated to 4°C, stabilized for 30 min, frozen in liquid nitrogen vapors (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were thawed and evaluated for sperm kinetics both by using computerized semen analysis with SCA® software (Sperm Class Analyzer version 5.0) and subjectively comparing specimens from the two animals using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique...

Influência do meio de conservação à base de água de coco em pó (ACP-102c) na manutenção da atividade mitocondrial de espermatozoides ovinos criopreservados / Influence of coconut powder water-based conservation medium (APC-102c) for maintaining mitochondrial activity of cryopreserved ram sperm

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders iskey for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is throughmicroscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and maynot provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to addreliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a moreaccurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10).Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS+ 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through therefrigeration curve up to 4°C (0.35°C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cmabove liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Bothfresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x),and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with theEosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained).Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3-diaminobenzidine(DAB); 200 sperms were counted, and classified into four...(AU)

Influência do meio de conservação à base de água de coco em pó (ACP-102c) na manutenção da atividade mitocondrial de espermatozoides ovinos criopreservados / Influence of coconut powder water-based conservation medium (APC-102c) for maintaining mitochondrial activity of cryopreserved ram sperm

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders iskey for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is throughmicroscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and maynot provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to addreliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a moreaccurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10).Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS+ 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through therefrigeration curve up to 4°C (0.35°C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cmabove liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Bothfresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x),and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with theEosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained).Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3’-diaminobenzidine(DAB); 200 sperms were counted, and classified into four...

Testicular morphological and ultrasonographic characterization of male gray brocket deers (Mazama gouazoubira) in different reproductive status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention.Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance ofmonitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotalcircumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocketdeer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods & Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicularvolume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes weresubjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD).Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity -EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen wasanalyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and threewere oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or KruskalWallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parametersusing Pearsons correlation for parametric variables and Spearmans correlation for non-parametric variables. F+ grouppresented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testescross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). Nosignificant differences were observed between the groups regarding...(AU)

Testicular morphological and ultrasonographic characterization of male gray brocket deers (Mazama gouazoubira) in different reproductive status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention.Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance ofmonitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotalcircumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocketdeer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods & Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicularvolume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes weresubjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD).Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity -EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen wasanalyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and threewere oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or KruskalWallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parametersusing Pearson’s correlation for parametric variables and Spearman’s correlation for non-parametric variables. F+ grouppresented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testescross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). Nosignificant differences were observed between the groups regarding...

Viabilidade espermática em sêmen in natura e refrigerado de veado-catingueiro / Sperm viability of brown brocket deer fresh and cooled sêmen

The brown brocket deer is in risk of extinction in two brazilian states. Therefore, more studies on semen evaluation may be useful to improve male reproductive capacity assessment. In this sense, this study evaluated the sperm viability using eosin-nigrosin and compared the results of fresh and cooled semen samples. Although the viability decreased in cooled semen, it maintained a satisfactory result.(AU)

Viabilidade espermática em sêmen in natura e refrigerado de veado-catingueiro / Sperm viability of brown brocket deer fresh and cooled sêmen

The brown brocket deer is in risk of extinction in two brazilian states. Therefore, more studies on semen evaluation may be useful to improve male reproductive capacity assessment. In this sense, this study evaluated the sperm viability using eosin-nigrosin and compared the results of fresh and cooled semen samples. Although the viability decreased in cooled semen, it maintained a satisfactory result.

Nova biotécnica de imunosexagem de espermatozoides de ruminantes / New biotech of immunosexing of ruminant’s spermatozoa

A sexagem espermática é uma biotecnologia que consiste na separação de espermatozoides portadores do cromossomo “X” ou “Y”. Esta técnica tem se destacado na área de produção animal, uma vez que tem despertado um grande interesse comercial, devido à possibilidade de aumentar a rentabilidade dos empreendimentos. Assim, esta revisão teve como objetivo abordar as principais técnicas utilizadas para sexagem espermática e seus avanços em relação a aplicabilidade no sistema pecuário. Todavia, diversos aspectos relacionados à técnica de sexagem de células espermáticas ainda necessitam ser elucidados, a fim de aprimorar ou desenvolver novas tecnologias que servirão para o desenvolvimento de metodologias mais eficientes e acessíveis aos produtores.

The sperm sexing is a biotechnology that consists of the separation of spermatozoa bearing the X or Y chromosome. This technique has been outstanding in animal production, since it has aroused a great commercial interest, due to the possibility of increasing the profitability of the enterprises. Thus, this review aimed to approach the main techniques used for sperm sexing and its advances regarding applicability in the livestock system. However, several aspects related to the sexing technique of sperm cells still need to be elucidated to improve or develop new technologies that will serve to develop more efficient and accessible methodologies for producers.