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The cooling of Litopenaeus vannamei shrimp spermatophores for assisted insemination can enable the transfer of gametes between reproduction laboratories. This study aimed to assess three extenders for cooling L. vannamei spermatophores for assisted insemination. Spermatophores were chilled at 15 °C for 24 or 48 hours using powdered coconut water ACP® (PCW), mineral oil (MO), and sterilized seawater (SSW) as extenders. All treatments demonstrated consistent responses over time. Apparent viability and morphological integrity percentages remained above 60% and 70%, respectively, across treatments and storage durations. Focusing on diluents, normal cell percentages for MO, SSW, and PCW treatments were 74.9±9.20%, 77.3±9.40%, and 78.1±6.35%, respectively, irrespective of storage time. The highest hatching rate was observed in the SSW treatment (80.67±12.01%), which was significantly superior to the PCW treatment (50.15±20.75%). The hatching rates observed in the MO treatment (71.47±18.83%) did not statistically differ from either PCW or SSW treatments. The cooling protocol successfully preserved the spermatophores' ability to maintain favorable levels of apparent viability, normal morphology, and hatching rates after 48 hours of storage at 15 °C using mineral oil, seawater, or ACP® as extenders. Sterilized seawater emerged as the most efficient diluent, delivering superior hatching rates following artificial insemination.(AU)

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Devido a contaminação bacteriana, utiliza-se antibióticos nos diluentes de sêmen para inibir o crescimento bacteriano durante a sua estocagem. Assim, este trabalho teve por objetivo determinar o limite de toxicidade de diferentes concentrações antibióticas da penicilina/estreptomicina adicionadas ao meio diluente do sêmen e investigar a influência sobre a motilidade espermática. Para isso, o sêmen de cinco reprodutores híbridos comerciais foi coletado uma vez por semana, durante nove semanas, através da técnica da mão enluvada. Cada ejaculado foi distribuído de forma igual entre todos os tratamentos, que consistiram em diferentes concentrações de solução de antibióticos, sendo: T1 = 0,0g/L (controle); T2 = 4,2g/L; e T3 = 12,6g/L. O sêmen diluído em água de coco em pó (ACP-103@) foi conservado por cinco dias e analisado nos dias: D0 (dia da coleta), D2 e D4 (último dia de conservação) quanto ao vigor e à motilidade espermática. Em D0, o sêmen conservado em diluente acrescido de 12,6g de solução antibiótica apresentou menor valor de vigor espermático em relação aos demais tratamentos (p<0,05). Já em D2 e D4, os tratamentos contendo antibióticos apresentaram resultados de vigor aquém dos obtidos no grupo controle (p<0,05). Em relação à motilidade espermática, os maiores percentuais de células móveis foram observados nas amostras de sêmen conservadas em ACP 0,0g/L (controle) quando comparados aos demais tratamentos (p<0,05). Conclui-se que as concentrações antibióticas utilizadas no estudo (4,2 e 12,6 g/L) mostraram efeitos tóxicos logo após a diluição do sêmen suíno, afetando de forma negativa parâmetros seminais durante a conservação a 17 °C.

Due to bacterial contamination, antibiotics are used in semen extenders to inhibit bacterial growth during storage. Thus, the present study aimed to determine the toxicity limit of different antibiotic concentrations of penicillin/streptomycin added to the semen extender and investigate the influence on sperm motility. For this purpose, semen from five commercial hybrid breeders was collected once a week for 9 weeks, using the gloved hand technique. Each ejaculate was distributed equally among all treatments, which consisted of different concentrations of antibiotic solution: T1 = 0.0g/L (control); T2 = 4.2g/L; T3 = 12.6g/L. The semen diluted in powdered coconut water (ACP-103@) was kept for 5 days and analyzed on the following days: D0 (collection day), D2, and D4 (last conservation day) for sperm vigor and motility. In D0, the semen preserved in the diluent added with 12.6 g of antibiotic solution showed a lower sperm vigor value compared to the other treatments (p<0.05). In D2 and D4, treatments containing antibiotics presented vigor results below those obtained in the control group (p<0.05). Regarding sperm motility, higher percentages of mobile cells were observed in semen samples conserved in ACP 0.0 g/L (control) when compared to the other treatments (p<0.05). It is concluded that the antibiotic concentrations used in the study (4.2 and 12.6 g/L) showed toxic effects soon after the swine semen, negatively affecting seminal parameters during storage at 17 °C.

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Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.(AU)

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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)

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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)

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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...

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The aim of this work went to evaluate the spermatic morphology of ram semen cooled to 4 °C in nature (INCW) and in powdered coconut water (ACP-102®) during the rainy and dry season in the Northeast of Brazil. The semen of four rams was collected, divided into two fractions and diluted in INCW and ACP-102®. The samples were conditioned in refrigerator to 4 °C and after 2, 24 and 48 hours of cooling were submitted at thermo resistant test (TT). Semen slides were executed in the beginning and in the end of TT to evaluation of the spermatic morphology (SM). The SM parameters, within different preservation times (2, 24 and 48h) and extenders (INCW and ACP-102®), were expressed in media and standard deviation (SD) and submitted to Tukey test (p<0.05). According to the diluted samples in ACP-102®, was observed a percentage increase of morphology normal spermatozoon in the rainy season as was verified in the dry season. In conclusion, the ACP-102® extender present good preserve capacity. Agreed with this study, the raining season did not have influence on the characteristics of spermatic morphology.

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Animal reproduction represents one of the most important factors, and the use of reproductive biotechnologies that help increase production is essential. The comet technique is the simple and quick way to detect pre-mutagenic lesions and assist in studies on environmental biomonitoring, toxicological genetics, biological radiation, DNA repair process and genetic ecotoxicology. The objective of this study was to evaluate the viability of DNA from sperm cells from goat semen submitted to the cryopreservation process in powdered coconut water (ACP101c/102c). The experiment was carried out at the Federal University of Piauí with two goats of reproductive age. The evaluation of the spermatic DNA integrity was performed using an immunofluorescence microscope. Classes from 0 to 3 were used, 0 being no damage and 3, the tail of the comet greater than twice the size of the nucleoid. The results obtained in the G2 test group (ACP 101c-102c) showed a slight tail formation, indicating a slight fragmentation of DNA. It was concluded that in the control group GC1 (TRIS + egg yolk of Gallu gallus domesticus) and experimental group (ACP 101c - 102c) there was no significant difference.

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The objective of this study was to test the efficiency of powdered coconut water (ACP-406®) base-medium without or with the addition of supplements on in vitro culture of isolated goat secondary follicles. Follicles were cultured for 18 days in α-MEM or in ACP-406®, both without supplements (referred to as α-MEM and ACP, respectively), or both supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred to as α-MEM+ and ACP+). Follicular morphology, antrum formation, follicular and oocyte diameter, levels of glutathione (GSH), and chromatin configuration after in vitro maturation were evaluated. At the end of culture, ACP-406® base-medium (without or with supplements) showed a higher (P < 0.05) percentage of normal follicles than α-MEM (without or with supplements). Antrum formation was similar among α-MEM+, ACP and ACP+, and significantly higher than α-MEM without supplements. The follicular diameter was greater in ACP+ than α-MEM, and similar to other treatments. Moreover, fully and daily grown rates were higher (P < 0.05) in ACP-406® base-medium (without or with supplements) than α-MEM (without or with supplements). Levels of GSH were similar between ACP+ and α-MEM+ treatments. Both ACP+ and α-MEM+ allowed meiotic resumption without a significant difference between the two groups. In conclusion, supplemented ACP-406® base-medium maintained follicular survival and promoted the development as well as meiotic resumption of isolated goat secondary follicles cultured in vitro for 18 days.

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This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 μg/mL penicillin and 100 μg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P 0.05) among treatments. Overall, all treatments had lower primordial follicles (P 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

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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...

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O objetivo deste trabalho foi avaliar a relação entre a concentração de proteínas no plasma seminal ovino com parâmetros de qualidade do sêmen criopreservado em diluentes TRIS e em Água de Coco em Pó (ACP102c). Um total de 12 colheitas/animal em ovinos Santa Inês (n=2) e Dorper (n=2) foram realizadas, para obtenção do plasma seminal e para criopreservação nos diluentes testados. Foi utilizado o método de Bradford para determinação da concentração de proteína. Para avaliação do sêmen fresco e criopreservado foram utilizados os testes de viabilidade por eosina-nigrosina e teste hiposmótico (HOST), além dos parâmetros de motilidade em sistema de análise de sêmen auxiliada por computador (CASA). Baseado no valor médio da concentração de proteína, os ejaculados foram agrupados em alta (AP) e baixa (BP) concentração proteica. Correlações significativamente positivas foram encontradas entre a concentração de proteína com os parâmetros motilidade progressiva (PROG) e congelabilidade (CONGEL) e negativa para deslocamento lateral de cabeça (ALH) nas amostras criopreservadas em ambos diluentes, além de correlação positiva para motilidade total (MOT) em TRIS. Sêmen de ejaculados do grupo AP criopreservados em TRIS apresentaram MOT, PROG e CONGEL significativamente superiores ao grupo BP e inferiores para ALH. Quando criopreservado em ACP102c, sêmen do grupo AP foi superior ao BP para PROG e CONGEL. Variações na qualidade do sêmen criopreservado entre ejaculados podem estar associados à concentração de proteínas do plasma seminal, e estas podem atuar na manutenção da progressividade de espermatozoides ovinos criopreservados em diluentes TRIS e ACP102c.

The objective of this study was to evaluate the relation between the protein concentration in ram seminal plasma and quality parameters of the semen cryopreserved in TRIS and Powdered Coconut Water (ACP102c) extenders. A total of 12 semen collections/animal of Santa Ines (n=2) and Dorper (n=2) rams were performed to obtain seminal plasma and semen cryopreservation in work extenders. The protein content was determined using the Bradford's method. The eosin-nigrosine and hyposmotic (HOST) viable tests and motility parameters obtained by computer assisted semen analysis were evaluated for fresh and cryopreserved semen. The ejaculates were grouped in high (AP) and low (BP) protein content, based on the mean value of protein concentration. Significantly positive correlations were found between the protein content and progressive motility (PROG), freezability (CONGEL) and negative for lateral head displacement (ALH) in the semen cryopreserved in both extenders. Therefore, positive correlation was found for total motility (MOT) in TRIS. Semen samples from AP group cryopreserved in TRIS extender were significantly better than BP group for MOT, PROG and CONGEL, and inferior for ALH. When cryopreserved in ACP102c, the AP group were better than BP group for PROG and CONGEL. Variations in the quality of cryopreserved semen from different ejaculates may be associated with the concentration of seminal plasma proteins and may act to maintain the progressiveness of ram sperm cryopreserved in TRIS and ACP102c extenders.

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This study aimed to evaluate the effect of powered coconut water as preservant of cocks semen in different times. The experimental method was completely randomized, with treatments constituted by different times (1, 5, 10 and 15 minutes) of powered coconut water as preservant of cocks semen (Rhode Island Red lineage with 30-weeks). After collect and use of powered coconut water as preservant, the semen samples were used for inseminated of breeders of same lineage and age (16 per treatment). 280 eggs were collected for evaluation of effects on chicks (70 eggs per treatment). Were evaluated the incubation yields, chick/egg relation and gastrointestinal tract development of chicks. The data collected were submitted for variance analysis and evaluated by Tukey test at 5% of significance. Differences (p 0.05) were observed in incubation yields, chick/egg relation and gastrointestinal tract development, with the exception (p>0.05) of oropharynx + oesophagus length and yolk sac weight. From these results, it was concluded that the powered coconut water can be used as preservation of cocks semen up to 15 minutes. Cells exposed more time to nutrients provided by the powdered coconut water showed eggs and chicks most heavier, and better results in the incubation yields and embryo development.

O objetivo com este estudo foi avaliar o efeito da água de coco como preservante do sêmen de galos em diferentes períodos. O delineamento experimental foi inteiramente casualizado, onde os tratamentos consistiam de diferentes períodos (1, 5, 10 e 15 minutos) de ação da água de coco como preservante do sêmen de galos (linhagem Rhode Island Red com 30 semanas). Após a coleta e submissão do sêmen a ação da água de coco, estas foram utilizadas para inseminação de 64 matrizes de mesma linhagem e idade (16 por tratamento). Foram coletados 280 ovos (70 ovos por tratamento) para avaliação dos efeitos da preservação do sêmen sobre a progênie. Foram avaliados os rendimentos de incubação, relação pinto/ovo e o desenvolvimento do trato gastrointestinal dos pintainhos. Os dados coletados foram submetidos à análise de variância e avaliados pelo teste de Tukey a 5% de significância. Diferenças (p 0,05) foram observadas nos resultados dos rendimentos de incubação, relação pinto/ovo e desenvolvimento do trato gastrointestinal, à exceção (p>0,05) dos resultados de comprimento da orofaringe + esôfago e peso do saco vitelino. A partir dos resultados obtidos, concluiu-se que a água de coco em pó pode ser utilizada como preservação do sêmen de galos até 15 minutos. As células expostas por mais tempo aos nutrientes da água de coco em pó proporcionaram ovos e pintos mais pesados, e melhores resultados nos rendimentos de incubação e desenvolvimento embrionário.

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Na conservação seminal é necessária a utilização de diluentes que forneçam nutrientes e protejam as células espermáticas contra o choque térmico. Os aditivos de origem animal, como a gema de ovo e o leite, amplamente empregados na preservação do sêmen, representam um risco potencial de contaminação. Com o intuito de reduzir esses impactos quanto ao uso de substâncias de origem animal, esta revisão teve como objetivo abordar os principais pontos acerca da utilização de diluentes para congelação do sêmen de pequenos ruminantes livres de gema de ovo, um diluente constituído 100% por produtos de origem vegetal.

For semen conservation, it is necessary to use extenders that provide nutrients and protect the spermatozoa against thermal shock. Animal source additives, such as egg yolk and milk, widely used in semen preservation, represent a potential contamination risk. To reduce impacts related to the use of animal origin substances, this review aimed to address the main points about the use of extenders for small ruminant sperm freezing free of egg yolk, an extender composed by 100% vegetable origin products.

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Este estudo foi realizado para determinar o efeito de hidroxitolueno butilado (BHT) sobre a qualidade do sêmen canino congelado e descongelado, utilizando o diluidor à base de água de coco em pó (ACP-106c). Para tanto, foram realizadas quinze coletas de sêmen provenientes de cinco cães. O sêmen obtido foi diluído em ACP-106c acrescido de glicerol e gema de ovo. As amostras foram então transferidas para tubos contendo diferentes concentrações de BHT (0; 0,5; 1,0 e 2,0 mM). Em seguida, as amostras foram envasadas, congeladas e armazenadas em nitrogênio líquido. O sêmen coletado foi avaliado in natura quanto aos seguintes parâmetros: coloração, volume da fração espermática, motilidade total, vigor, concentração, morfologia e funcionalidade de membrana espermática. Após uma semana, as amostras foram descongeladas e avaliadas por meio de análise computadorizada, como também foram realizadas análises da funcionalidade de membrana e da morfologia espermática. A motilidade progressiva no grupo BHT 2,0 mM foi significativamente superior (P < 0,05) do que a do grupo BHT 0 mM (27,6 ± 11,7% vs. 19,0 ± 9,5%, respectivamente). Em todos os demais parâmetros avaliados, não houve diferença entre os grupos testados. Portanto, conclui-se que a adição do BHT ao diluidor ACP-106c não afetou a qualidade do sêmen canino pós-descongelação.

This study was conducted to determine the effect of butylated hydroxytoluene (BHT) on the quality of canine sperm frozen and thawed using the powdered coconut water based (ACP-106c) extender. Therefore, fifteen ejaculates were collected from five dogs. Semen obtained was diluted in ACP-106c added of glycerol and egg yolk. The samples were then transferred to tubes containing different concentrations of BHT (0, 0.5, 1.0 and 2.0 mM). After that, the samples were filled into straws, frozen and stored in liquid nitrogen. Fresh semen was evaluated for the following parameters: color, sperm fraction volume, total motility, vigor, concentration, morphology, and HOST test. After one week, the samples were thawed and evaluated by computer analysis, as well as for membrane functionality and sperm morphology. Progressive motility in the 2.0 mM BHT group was significantly higher (P <0.05) than that of the 0 mM BHT group (27.6 ± 11.7% vs. 19.0 ± 9.5%, respectively). Regarding all other parameters evaluated, there was no difference between the groups tested. Therefore, the addition of BHT to the ACP-106c extender did not affect the quality of canine semen after thawing.

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Durante o pré-parto e o início da lactação, as ovelhas deverão suportar um gigantesco aumento na demanda energética, devido ao desenvolvimento final do feto, produção de colostro e leite pela glândula mamária. Essa demanda estimula mobilização de gorduras do tecido adiposo. No entanto, uma mobilização excessiva pode induzir um desequilíbrio na metabolização hepática de carboidratos e gorduras, o que pode resultar em desequilíbrios metabólicos que desencadeiam doenças pré-parto e falhas nos processos reprodutivos após o parto. A adição de água de coco em pó (ACP) no período de 110 dias gestacionais até 60 dias pós parto em ovelhas, pode gerar mudanças metabólicas nos níveis plasmáticos de glicose, séricos de AGNE (ácidos graxos não esterificados) e BHB (beta-hidroxibutirato). O ACP é rico em hormônios promotores de crescimento e agentes antiiflamatórios, podendo neutralizar os efeitos negativos do balanço energético negativo. O objetivo deste estudo foi avaliar quais os efeitos da adição do ACP têm sobre os valores plasmáticos de glicose, e séricos de BHB e NEFA de ovelhas gestantes, e a influência no ganho de peso do borrego. As ovelhas gestantes foram distribuídas em dois grupos. Fêmeas (n= 5) do grupo teste (GT) receberam uma dose oral diária de 30g de água de coco em pó associada ao concentrado. Essa dose foi administrada entre o 110° dia de gestação (M0) até o 60° dia após o parto. Exames laboratoriais foram efetuadas nos seguintes dias: no dia do parto e nos dias 7, 21, 30 e 60 após o parto. Foi possível observar que o ACPg (grupo teste) permaneceu estável com os animais mantendo os níveis de glicose sem apresentar alterações, mesmo no dia do parto, apesar de 80% das ovelhas estarem gestante de gêmeos. Os níveis de BHB e NEFA também foram melhores em comparação ao Cg (grupo controle). O Cg apresentou maior instabilidade ao longo do experimento, com momentos de hiper e hipoglicemia, aumento do valor do fibrinogênio em alguns animais, BHB e NEFA também apresentaram alterações. Cordeiros da ACPg apresentaram melhor ganho de peso em relação ao Cg. Concluiu-se que o estado energético das ovelhas com gestação gemelar suplementadas com ACP® foi superior ao grupo não suplementado, o que pode interferir positivamente no balanço energético negativo.

During prepartum and early lactation, ewes will have to withstand a gigantic increase in energy demand, due to the final development of the fetus, production of colostrum and milk by the mammary gland. This demand stimulates mobilization of fats from adipose tissue. However, excessive mobilization can induce an imbalance in the hepatic metabolism of carbohydrates and fats, which can result in metabolic imbalances that trigger antepartum diseases and failures in the reproductive processes after delivery. The addition of powdered coconut water (PCA) in the transition period in sheep can generate metabolic changes in the serum levels of glucose, NEFA (non-esterified fatty acids) and BHB (betahydroxybutyrate). ACP is rich in growth-promoting hormones and antiinflammatory agents, which can counteract the negative effects of negative energy balance. For this, we evaluated the effects of the addition of ACP on the serum values of glucose, BHB, NEFA and fibrinogen in pregnant ewes, and the influence on the weight gain of the lamb. Pregnant ewes were divided into two groups. Females in the test group (TG) received a daily oral dose of 30g of powdered coconut water associated with the concentrate. This dose was administered between the 110th day of gestation (M0) until the 60th day after delivery. Laboratory tests were performed on the following days: 110th day of pregnancy (before starting the administration of the product, on the day of delivery and on days 7, 21, 30 and 60 after delivery. It was possible to observe that the ACPg (test group ) remained stable with the animals maintaining glucose levels unchanged, even on the day of parturition, despite the fact that 80% of the ewes were pregnant with twins. BHB and NEFA levels were also better compared to Cg (control group). Cg showed greater instability throughout the experiment, with moments of hyper and hypoglycemia, increased fibrinogen value in some animals, BHB and NEFA also showed changes. ACPg lambs showed better weight gain in relation to Cg. the energy status of ewes with twin pregnancy supplemented with ACP® was higher than the non-supplemented group, which can positively interfere in the negative energy balance.

Resumo

For artificial insemination, it is essential to use frozen semen, however the freezing process causes deleterious changes to the structure and integrity of sperm membranes that compromise the function of sperm. To avoid this cellular damage, extenders and suitable substrates must be used to recover the highest possible number of viable cells post-thaw. To this end, in the first experiment, we evaluated three different extenders: TES-TRIS, which is widely used for buffaloes; and an extender composed of powdered coconut water-based (ACP-112®) with or without milk (ACP-112®-milk) for buffalo semen freezing. In the second experiment, we evaluated the effect of Lippia origanoides oil extract on protecting buffalo sperm against cryoinjury arising from freezing semen. Semen was collected from ten buffalo bulls (10 ejaculates/bull) and diluted in TES-TRIS (control), ACP-112® or ACP-112®-Milk in the first experiment. In the second experiment, the samples were diluted in the diluent with the best results for sperm quality obtained in experiment I, and 2.5 μg mL-1, 5 μg mL-1 or 10 μg mL-1 of the plant extract was added to treatments; and a control group containing only the diluent was also included. The fresh semen was analyzed for conventional features such as motility, concentration, morphology and viability. After thawing, the samples were evaluated again for motility, vigor and supra-vital staining, and then, were performed the of thermal-resistance test, hypoosmotic test and evaluated sperm membrane integrity with the fluorescent probes PI, FITC-PSA and JC-1 using flow cytometry. The data were submitted to ANOVA, and the results were compared by Tukey’s test at a significance of 5%.

Para a implantação da inseminação artificial é indispensável à utilização de sêmen congelado, que pode provocar mudanças deletérias na estrutura e na integridade das membranas espermáticas, comprometendo sua função. Para evitar estes danos celulares, há a necessidade de se utilizar meios diluidores e substratos adequados que recuperem o maior número possível de células viáveis pós-descongelação. Para isso, foram avaliados, no experimento I, três diferentes diluidores, o diluidor TES-TRIS, bastante utilizado para bubalinos, e um diluidor a base de água de coco em pó (ACP-112), associado ou não ao leite (ACP-112-Leite), na congelação do sêmen de bubalinos; e no experimento II, foi avaliado o efeito do óleo extraído da Lippia origanoides na proteção dos espermatozóides contra as crioinjúrias decorrentes da congelação do sêmen bubalino. Foram utilizados 10 touros bubalinos para as colheitas de sêmen (10 ejaculados/touro), sendo os ejaculados diluídos em TES-TRIS (controle), ACP-112 e ACP-112-Leite no experimento I; e no experimento II, os ejaculados foram diluídos no melhor diluidor obtido no experimento I, acrescido de 2.5 μg mL-1, 5 μg mL-1 e 10 μg mL-1 da planta e o grupo controle, constituído somente do diluidor. O sêmen recém colhido foi analisado quanto as características convencionais, tais como, motilidade, concentração, morfologia e viabilidade. Após a descongelação das amostras foram avaliados novamente, motilidade e viabilidade espermática, e posteriormente, foram realizados os testes de termo-resistência, hiposmótico e de avaliação das membranas dos espermatozóides, através das sondas fluorescentes PI, FITC-PSA e JC-1, utilizando a citometria de fluxo. Os dados obtidos foram submetidos à ANOVA e ao Teste de Tukey a 5%.

Resumo

O objetivo da presente pesquisa foi alcançado com a divisão da pesquisa em dois experimentos: (1) aperfeiçoar o protocolo de congelação utilizando água de coco em pó (ACP-104) como diluente para a criopreservação seminal de carpa comum; (2) avaliar o efeito da suplementação das vitaminas C (ácido ascórbico) ou E (α-tocoferol) sobre os melhores diluidores testados no experimento 1 na qualidade do sêmen pós-descongelado da espécie. Para o experimento 1, foram formados oito pools de sêmen, provenientes de 14 machos selecionados. As amostras seminais coletadas foram avaliadas quanto à motilidade total, à velocidade, ao percentual de espermatozoides normais e à vitalidade espermática antes e depois da criopreservação seminal. Esta foi realizada em meio ACP-104 acrescido de dimetilsulfóxido (DMSO), ou etilenoglicol (EG), ou glicerol, ou metanol, todos à concentração de 10%, diluídos em 1:3 (sêmen:diluidor). As amostras foram, então, congeladas em vapor de nitrogênio líquido em dry shipper e estocadas em nitrogênio líquido (-196°C). Para o experimento 2, foram formados oito pools provenientes da coleta de sêmen de 15 machos. As amostras seminais foram avaliadas seguindo as mesmas análises do experimento 1, acrescentando-se a duração da motilidade total. A criopreservação seminal utilizou-se do meio ACP-104 acrescido de DMSO ou EG, suplementado ou não com vitamina C ou E. Os melhores resultados encontrados no experimento 1 foram obtidos com o DMSO e o EG. Estes não diferiram significativamente entre si para a motilidade total (24% e 28%; P>0,05) e a normalidade espermática (32% e 26%; P>0,05), respectivamente. Para o experimento 2, o EG suplementado com vitamina E produziu significativamente resultados superiores de motilidade total, normalidade espermática e duração da motilidade em relação ao DMSO, concluindo-se que o EG deve ser, portanto, o crioprotetor de escolha a ser utilizado com o ACP-104 suplementado ou não com vitamina E.(AU)

The objective was achieved by dividing the research into two experiments: (1) improving the freezing protocol using powdered coconut water (ACP-104) as a diluent for the cryopreservation seminal of common carp; (2) evaluating the effect of supplementation of vitamins C (ascorbic acid) or vitamin E (α-tocoferol) with the best extenders tested in experiment 1 on the quality of post-thawed. For experiment 1, semen pools from 14 selected males were formed. Seminal samples were evaluated for total motility, velocity, percentage of normal sperm and sperm vitality before and after the seminal cryopreservation. This was done in ACP-104 extender plus dimethyl sulfoxide (DMSO), or ethylene glycol (EG), or glycerol or methanol all at concentration 10% diluted in 1:3 (semen:extender). The samples were frozen in vapors of nitrogen into dry shippers and stored in liquid nitrogen (-196 °C). For experiment 2, eight pools were formed from the 15 males. The semen samples were evaluated following the same analysis of experiment 1 adding duration of total motility. The sperm cryopreservation was performed in extenders ACP-104 plus DMSO or EG supplemented or not with vitamin C or E. The best results found in Experiment 1 were obtained with DMSO and EG. They do not differ significantly for total motility (24% and 28%; P>0.05) and normal sperm (32% and 26%; P>0.05) respectively. For experiment 2, EG supplemented with vitamin E, produced significantly better results overall motility, sperm normality and duration of motility relative to DMSO. In conclusion, EG should be the cryoprotectant of choice for use with the ACP-104 supplemented or not with vitamin E.(AU)

Resumo

The study aimed to evaluate the mitochondrial activity of cryopreserved ram sperm preserved inmedium based on powdered coconut water (ACP-102c). Five sperm collections of two rams were held. Eachejaculate was divided into two treatments: T1 (ACP-102c+egg yolk+glycerol) and T2 (TRIS+eggyolk+glycerol). After dilution, the samples were filled into 0.5 ml straws, chilled to 4 °C and stabilized for 2 h.They were then frozen and stored in liquid nitrogen at -196°C. The activity mitochondrial of treatments wereevaluated in time 0h and post-thawing. Data were expressed as mean ± standard deviation and submitted to theShapiro-Wilk normality test and subsequently suffered angular transformation into arcsine of percentages. Theresults were considered significant when P < 0.05. There was no significant difference between treatments. Itwas concluded that ACP-102c was able to maintain mitochondrial activity pot-thawing.

Resumo

The conservation of boar semen at lower temperatures might contribute to the further expansion of artificial insemination in this species. Egg yolk cryoprotectant properties have already been extensively tested on sperm cryopreservation of several species. This study aimed to test different temperature curves for the conservation of boar semen diluted with coconut milk powdered (ACP®-103) add 7% egg yolk and to verify which one better maintains sperm viability. For this, 36 ejaculates were diluted and stored at 17, 10 and 5 C. Daily analysis of vigor and motility were performed, and on days D0, D2, and D4 semen was evaluated regarding vitality, morphology, and osmotic resistance. For the statistical analysis we performed the tests of Kruska-Wallis with Dunns post-test (nonparametric data) and ANOVA and Tukey test (parametric data). The storage temperature of 10 C was the best one   to maintain spermatic motility at appropriate levels to be used in an artificial insemination program. Analyses of viability, morphology, and hypoosmotic test did not show statistical difference among the treatments. In conclusion, the best temperature curve was 10 C with diluted semen previously kept at 17 C to maintain the viability of sperm cells in pigs for a longer period.

A conservação do sêmen suíno em temperaturas mais baixas pode permitir uma maior expansão    da inseminação artificial nessa espécie. A gema de ovo apresenta propriedades crioprotetoras já amplamente testadas na conservação seminal de diversas espécies. Este trabalho teve por objetivo testar diferentes curvas de temperatura na conservação do sêmen suíno diluído em água de coco    em pó (ACP®-103) acrescido de 7% de gema de ovo e verificar qual delas mantém por mais tempo  a viabilidade espermática. Para tanto, o sêmen de 36 ejaculados foi diluído e conservado a 17,        10 e 5 C. Diariamente foram realizadas análises de vigor e motilidade e nos dias D0, D2 e D4 o sêmen foi avaliado quanto à sua viabilidade, morfologia acrossomal e resistência osmótica. Para a análise estatística foram utilizados os testes de Kruska-Wallis, com pós-teste de Dunns (dados não paramétricos) e Anova com teste de Tukey (dados paramétricos). A conservação à temperatura de 10 C foi a que melhor manteve o vigor espermático e a motilidade em níveis adequados para ser utilizado em um programa de inseminação artificial. As análises de vitalidade, morfologia e teste hiposmótico não apresentaram diferença estatística entre os tratamentos avaliados. Em conclusão, a melhor curva de temperatura foi a de 10 C com sêmen diluído por manter por um período maior a viabilidade da célula espermática suína.