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The present study evaluated the cryoprotectant efficacy of dimethylacetamide (DMA) and ethylene glycol in a one-step protocol to freeze boar sperm. The sperm-rich portion of the ejaculates from two boars were collected once a week, for 10 weeks. After collection, the ejaculates were diluted (1:1; v/v) in the cooling extender. After determining their spermatozoa concentration, the ejaculates were pooled with the same number of spermatozoa from each boar and stabilized at 20°C for 120 min. Distinct cryoprotectants were added to the cooling extender at 20 °C, at different concentrations, composing six treatments: 1.25% and 2.5% glycerol (control); 1.25% and 2.5% ethylene glycol; 2.5% and 5.0% DMA. The samples were stored in 0.25 mL straws, containing 35 × 106 spermatozoa. After 90 min at 20 °C, the straws were submitted to a cooling curve until 5 °C (0.3 to 0.5 °C/min) and kept at 5°C for 60 min. Freezing was conducted by placing the straws horizontally 5 cm above the liquid nitrogen for 10 min, followed by immersion on liquid nitrogen. After thawing at 37 °C for 30 seconds, sperm quality was evaluated through a computer-assisted semen analysis system and flow cytometry. Sperm motility was greater (P< 0.05) in treatments with 5.0% and 2.5% DMA (22.2 ± 2.6% and 20.0 ± 2.8%, respectively) than in treatment with 2.5% ethylene glycol (8.2 ± 1.0%). The integrity of the plasma membrane (P = 0.08) and mitochondrial membrane potential (P = 0.27) was similar among the treatments. The treatment with 2.5% ethylene glycol was the least efficient to maintain intact acrosome membrane (P< 0.01). Some kinetics parameters (DAP, DCL, DSL, VAP, VCL, VSL e ALH) were positively affected by 5.0% DMA. The one-step freezing protocol resulted in unsatisfactory boar sperm motility after thawing, regardless of the cryoprotectant.

O presente estudo objetivou avaliar a eficácia de um protocolo one-step de congelamento do sêmen suíno utilizando dimetilacetamida (DMA) e etilenoglicol como crioprotetores. Durante 10 semanas, a fração rica dos ejaculados de dois machos suínos foram coletados, uma vez por semana. Após a coleta, os ejaculados foram diluídos (1:1; v/v) no diluidor de resfriamento. Após a avaliação da concentração espermática, os ejaculados foram agrupados em um pool com o mesmo número de espermatozoides de cada macho e estabilizados a 20 °C por 120 min. Os criopropetores foram adicionados ao diluidor de congelamento a 20 °C, em diferentes concentrações, compondo seis tratamentos: glicerol (controle), 1,25% e 2,5%; etilenoglicol, 1,5% e 2,5%; e DMA, 2,5% e 5,0%. As amostras foram armazendadas em palhetas de 0,25 mL contendo 35 x 106 espermatozoides. Após 90 min a 20 °C as palhetas foram submetidas a uma curva de resfriamento até 5 °C (0,3 a 0,5 °C/mim) e mantidas a 5 °C por 60 min. O congelamento foi realizado a partir da colocação das palhetas horizontalmente a 5 cm acima do nitrogênio líquido por 10 min, com sua posterior imersão no nitrogênio líquido. Após o descongelamento a 37 °C por 30 segundos a qualidade espermática foi avaliada através de um sistema computadorizado e por citometria de fluxo. A motilidade espermática foi maior (P < 0,05) nos tratamentos com 5,0% e 2,5% DMA (22,2 ± 2,6% e 20,0 ± 2,8%, respectivamente) do que no tratamento com 2,5% etilenoglicol (8,2 ± 1,0%). A integridade da membrana plasmática (P = 0,08) e potencial de membrana mitocondrial (P = 0,27) foi similar entre os tratamentos. O tratamento com 2,5% de etilenoglicol foi menos eficiente em manter membrana acrossomal intacta (P< 0,01). Alguns parâmetros de cinética espermática (DAP, DCL, DSL, VAP, VCL, VSL e ALH) foram afetados positivamente pelo uso de DMA a 5.0%. O protocolo simplificado para congelamento de sêmen suíno resultou em motilidade espermática insatifatória após o descongelamento, independente do crioprotetor utilizado.

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Treatment with prostaglandin F2α (PGF) induces ovulation and increases conception rates in cows, while improving embryo production in buffalos. However, its effect on superovulated cows is unknown. This study verified whether single PGF administration concurrent with artificial insemination (AI) improves fertilization and embryo production rates in superovulated cows. In each replicate, embryo donor cows were equally allocated to two groups: the untreated control and PGF groups. The latter of which received 482 µg of cloprostenol concurrent with the first AI. Each cow (n = 35) was subjected to two superovulations (SOV) in a crossover design (total = 70 embryo collections). In the control and PGF groups, respectively, the observed responses were [median (95% CI)]: 12 (10-18) and 15 (12-18) total structures, 9 (7-11) and 7 (6-10) viable embryos, 1 (0-1) and 1 (1-3) degenerated embryos, and 1 (0-3) and 2 (0-5) oocytes (P > 0.05). In conclusion, single PGF treatment concurrent with the first AI did not affect embryo production in superovulated cows.

A prostaglandina F2α (PGF) pode induzir a ovulação e melhorar tanto a concepção em vacas, como a produção de embriões em búfalas, mas o efeito em vacas superovuladas é desconhecido. Esse estudo teve como objetivo verificar se a administração de uma dose de PGF na inseminação artificial (IA) após a superovulação (SOV) melhora as taxas de fecundação e produção embrionária em vacas. Em cada replicação, vacas doadoras de embriões foram equilibradamente alocadas em dois grupos: controle, não tratado, ou PGF, que recebeu 482 µg de cloprostenol no momento da primeira IA. Cada doadora (n = 35) foi submetida a duas SOV em um delineamento crossover (total = 70 coletas de embriões). Nos grupos controle e PGF, respectivamente, foram observados [medianas (IC 95%)]: 12 (10-18) e 15 (12-18) estruturas totais; 9 (7-11) e 7 (6-10) embriões viáveis; 1 (0-1) e 1 (1-3) embriões degenerados; e 1 (0-3) e 2 (0-5) ovócitos (P > 0,05). Conclui-se que uma única administração de PGF no momento da primeira IA não afeta a produção embrionária de vacas superovuladas.

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ABSTRACT: Treatment with prostaglandin F2α (PGF) induces ovulation and increases conception rates in cows, while improving embryo production in buffalos. However, its effect on superovulated cows is unknown. This study verified whether single PGF administration concurrent with artificial insemination (AI) improves fertilization and embryo production rates in superovulated cows. In each replicate, embryo donor cows were equally allocated to two groups: the untreated control and PGF groups. The latter of which received 482 µg of cloprostenol concurrent with the first AI. Each cow (n = 35) was subjected to two superovulations (SOV) in a crossover design (total = 70 embryo collections). In the control and PGF groups, respectively, the observed responses were [median (95% CI)]: 12 (10-18) and 15 (12-18) total structures, 9 (7-11) and 7 (6-10) viable embryos, 1 (0-1) and 1 (1-3) degenerated embryos, and 1 (0-3) and 2 (0-5) oocytes (P > 0.05). In conclusion, single PGF treatment concurrent with the first AI did not affect embryo production in superovulated cows.

RESUMO: A prostaglandina F2α (PGF) pode induzir a ovulação e melhorar tanto a concepção em vacas, como a produção de embriões em búfalas, mas o efeito em vacas superovuladas é desconhecido. Esse estudo teve como objetivo verificar se a administração de uma dose de PGF na inseminação artificial (IA) após a superovulação (SOV) melhora as taxas de fecundação e produção embrionária em vacas. Em cada replicação, vacas doadoras de embriões foram equilibradamente alocadas em dois grupos: controle, não tratado, ou PGF, que recebeu 482 µg de cloprostenol no momento da primeira IA. Cada doadora (n = 35) foi submetida a duas SOV em um delineamento crossover (total = 70 coletas de embriões). Nos grupos controle e PGF, respectivamente, foram observados [medianas (IC 95%)]: 12 (10-18) e 15 (12-18) estruturas totais; 9 (7-11) e 7 (6-10) embriões viáveis; 1 (0-1) e 1 (1-3) embriões degenerados; e 1 (0-3) e 2 (0-5) ovócitos (P > 0,05). Conclui-se que uma única administração de PGF no momento da primeira IA não afeta a produção embrionária de vacas superovuladas.

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The high lipid content in porcine oocytes impairs in vitro embryo production (IVP). Here, we evaluated the influence of two different lipid modulators during in vitro maturation (IVM) on the embryo development and the lipid content of oocytes and embryos. In Experiment I, oocytes were exposed to 50 µM docosahexaenoic acid (DHA) with (+) or without (-) the presence of porcine follicular fluid (pFF). In Experiment II, phenazine ethosulfate (PES) was added during IVM at two concentrations (0.5 and 0.05 µM). The pFF- with 50 µM DHA treatment impaired nuclear maturation, cleavage and blastocyst rates (p < 0.05). Oocytes in pFF- media accumulated less lipids (p < 0.05). The addition of 0.5 µ M PES reduced all development rates (p < 0.05) and resulted in higher lipid content for oocytes and embryos. Only 0.05 µM PES oocytes matured similarly to the control (p > 0.05), although embryo development and embryo lipid content was similar to 0.5 µM PES oocytes (p > 0.05). Thus, 50 µM DHA supplementation in the IVM medium without pFF impaired oocyte maturation and embryo development rates without interfering in oocyte lipid content even in the presence of pFF. Maturation with PES neither favored porcine embryo development nor reduced their lipid content.

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Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos <300 µm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are >300 µm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.(AU)

Métodos de criopreservação de embriões têm sido utilizados com diversos objetivos. Maiores taxas de coleta embrio-nária em éguas com lavagem uterina realizada 7 a 8 dias pós ovulação. A criopreservação de embriões equinos com diâmetro <300 µm (6-6,5 dias após a ovulação) permite a obtenção de taxas de prenhez satisfatórias. Embriões com diâmetro >300 µm (7º dia pós-ovulação) somente são adequadamente criopreservados quando submetidos a colabamento da blastocele. Objetivando avaliar a viabilidade da punção da blastocele com equipamento de baixa sofisticação e custo, 22 embriões coletados no 8º. dia pós-ovulação (D8) foram alocados aos seguintes grupos: (1) micropunção com uma agulha 30 G assistida por micromanipula-dor antes da vitrificação (n=4); (2) microssecção manual por lâmina antes da vitrificação (n=6); (3) sem manipulação anterior à vitrificação (n=8); e (4) transferidos a fresco (n=4). Apesar de altas taxas de reexpansão após a criopreservação, os embriões manipulados previamente a vitrificação não resultaram em prenhez aos 25 dias. Tanto os embriões não micromanipulados, quanto os transferidos a fresco resultaram em prenhezes aos 25 dias. A microssecção manual não se mostrou eficiente como método para aumento da criotolerância de embriões grandes, necessitando um aprimoramento visando a obtenção de prenhezes.(AU)

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Associations of the activity of the paraoxonase 1 (PON1) enzyme with boar sperm quality still needs to be characterized, since boar ejaculates present distinct portions with differences in sperm concentration and quality. This study evaluated PON1 activity in the serum, in the distinct portions of boar ejaculates and estimated correlations with sperm quality parameters. Ejaculates and blood samples were collected from six boars for three weeks (two per week per boar; n = 36). Serum and post-spermatic portion PON1 activities were positively correlated (P = 0.01) but were both uncorrelated with the PON1 activity in the sperm-rich portion and in the whole ejaculate (P > 0.05). Differences in PON1 activity among boars were only observed in the sperm-rich portion of the ejaculate (P < 0.05). The PON1 activity in the serum and in the post-spermatic portion was generally negatively correlated with parameters of spermatozoa kinetics (P < 0.05). In the sperm-rich portion, PON1 activity was positively correlated with sperm concentration (P < 0.0001), curvilinear distance and velocity (both P < 0.05) and DNA integrity (P < 0.05), but negatively correlated with straightness and linearity (P < 0.05). Thus, boar ejaculates with increased PON1 activity in the sperm-rich portion may present increased concentration and spermatozoa with acceptable curvilinear velocity and distance and DNA integrity, which suggests that PON1 activity may be a biomarker for potential fertility.(AU)

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Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos <300 µm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are >300 µm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day  8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in incr

RESUMO

Diversos hormônios podem ser utilizados em protocolos que visam otimizar biotécnicas reprodutivas como a inseminação artificial (IA) e inseminação artificial em tempo fixo (IATF), que exigem controle preciso do ciclo estral e da ovulação. Alguns hormônios possuem ação central no eixo hipotalâmico-hipofisário, como o hormônio liberador de gonadotrofinas (GnRH) e os ésteres de estradiolcomo o benzoato (BE) e o cipionato de estradiol (CE), enquanto outros como a gonadotrofina coriônica humana (hCG) e o hormônio luteinizante (LH) apresentam ação local, a nível ovariano. Atualmente os protocolos mais utilizados para indução da ovulação utilizam GnRH e estrógenos. Recentemente, foi demonstrado que as prostaglandinas possuem efeitos positivos na indução do processo ovulatório, mas seu mecanismo de ação não está totalmente esclarecido. A presente revisão tem por objetivo abordar os principais indutores da ovulação utilizados em protocolos para controle do ciclo estral em bovinos, relatando o período de ocorrência das ovulações após a sua aplicação.(AU)

Several hormones may be used in protocols aimed to optimize reproductive biotechniques such as artificial insemination (AI) and fixed-timed artificial insemination (FTAI) which require precise control of the estrous cycle and the ovulation. Some hormones act on the hypothalamic-pituitary axis, such as the gonadotrophin releasing hormone (GnRH) and estradiol esters such as estradiol benzoate (EB) and cypionate (EC), whereas others act directly on the ovaries, such as human chorionic gonadotrophin (hCG) and luteinizing hormone (LH). Currently, the most commonly used protocols for ovulation induction use GnRH or estrogen. Prostaglandins have recently been shown to have positive effects on induction of the ovulatory process, but their mechanism of action is not fully understood. The present review aims to address the main ovulation inducers used in protocols to control the estrous cycle in cattle, emphasizing the period between their administration and the occurrence of ovulation.(AU)

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Diversos hormônios podem ser utilizados em protocolos que visam otimizar biotécnicas reprodutivas como a inseminação artificial (IA) e inseminação artificial em tempo fixo (IATF), que exigem controle preciso do ciclo estral e da ovulação. Alguns hormônios possuem ação central no eixo hipotalâmico-hipofisário, como o hormônio liberador de gonadotrofinas (GnRH) e os ésteres de estradiolcomo o benzoato (BE) e o cipionato de estradiol (CE), enquanto outros como a gonadotrofina coriônica humana (hCG) e o hormônio luteinizante (LH) apresentam ação local, a nível ovariano. Atualmente os protocolos mais utilizados para indução da ovulação utilizam GnRH e estrógenos. Recentemente, foi demonstrado que as prostaglandinas possuem efeitos positivos na indução do processo ovulatório, mas seu mecanismo de ação não está totalmente esclarecido. A presente revisão tem por objetivo abordar os principais indutores da ovulação utilizados em protocolos para controle do ciclo estral em bovinos, relatando o período de ocorrência das ovulações após a sua aplicação.

Several hormones may be used in protocols aimed to optimize reproductive biotechniques such as artificial insemination (AI) and fixed-timed artificial insemination (FTAI) which require precise control of the estrous cycle and the ovulation. Some hormones act on the hypothalamic-pituitary axis, such as the gonadotrophin releasing hormone (GnRH) and estradiol esters such as estradiol benzoate (EB) and cypionate (EC), whereas others act directly on the ovaries, such as human chorionic gonadotrophin (hCG) and luteinizing hormone (LH). Currently, the most commonly used protocols for ovulation induction use GnRH or estrogen. Prostaglandins have recently been shown to have positive effects on induction of the ovulatory process, but their mechanism of action is not fully understood. The present review aims to address the main ovulation inducers used in protocols to control the estrous cycle in cattle, emphasizing the period between their administration and the occurrence of ovulation.

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Background: Escherichia coli (E. coli) is an enteropathogen that commonly causes diarrhea in calves. However, not all E. coli isolates are pathogenic. The aim of this study was to identify E. coli virulence factors derived from fecal samples collected in the southern region of Rio Grande do Sul state (RS) from calves with and without diarrhea, as well as investigate the antimicrobial susceptibility of E. coli isolates from calves with diarrhea. Materials, Methods & Results: Forty stool samples were collected in 12 farms, each one from calves having one day to six months of age, with and without diarrhea. The total DNA of from these isolates was extracted and a PCR using primers specific for the virulence factors Stx1, Eae, F41, F5 and STa was conducted. The susceptibility testing used the disk diffusion method and the susceptibility profile was evaluated against the following antimicrobials: ampicillin, penicillin, chloramphenicol, enrofloxacin, gentamicin, trimethoprim, sulfonamide, tetracycline and streptomycin. From all calves, 15 (15/40, 37.5%) had diarrheal stools and 25 (25/40, 62.5%) had normal or semi-liquid stools. Twelve (12/40; 30%) E. coli isolates showed at least one virulence factor. These factors were found in four isolates (4/15; 26.6%) from diarrheal stools and eight isolates (8/25; 28.5%) from normal stool. The Stx1 factor was identified in five isolates (5/40; 12.5%), and the Eae and the Sta factors in one (1/40; 0.2%) and in atypical associations between Stx1 and Eae and also between Eae and F41 in two isolates (2/40; 0.5%). Also, the Eae and Sta factors were identified in one isolate (1/40; 0.2%). The susceptibility test showed resistance to penicillin and tetracycline in 93% and 80% of the tested isolates, respectively. Discussion: The identification of virulence factors is necessary because E. coli is an enterobacterium present in calves gastrointestinal tract, to prove its pathogenicity. […](AU)

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