Oviductal extracellular vesicles miRNA cargo varies in response to embryos and their quality.

BACKGROUND: Increasing evidence points to an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. However, it remains unclear whether oEVs contribute to the recognition of the presence of embryos and their quality in the oviduct. Hence, we examined whether the molecular cargo of oEVs secreted by bovine oviduct epithelial cells (BOEC) differs depending on the presence of good (≥ 8 cells, G) or poor (< 8 cells, P) quality embryos. In addition, differences in RNA profiles between G and P embryos were analyzed in attempt to distinguish oEVs and embryonic EVs cargos. METHODS: For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53 h post fertilization as follows: BOEC with G embryos (BGE); BOEC with P embryos (BPE); G embryos alone (GE); P embryos alone (PE); BOEC alone (B) and medium control (M). After 24 h of co-culture, conditioned media were collected from all groups and EVs were isolated and characterized. MicroRNA profiling of EVs and embryos was performed by small RNA-sequencing. RESULTS: In EVs, 84 miRNAs were identified, with 8 differentially abundant (DA) miRNAs for BGE vs. B and 4 for BPE vs. B (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value < 0.01). CONCLUSIONS: These results indicated that oEVs are involved in the oviductal-embryo recognition and pointed to specific miRNAs with signaling and supporting roles during early embryo development.

Mitoquinone does not improve sperm cryo-resistance in bulls.

Oxidative stress is associated with impaired post-thaw sperm quality. As mitochondria are the main source of reactive oxygen species (ROS) in sperm, the goal of this study was to evaluate effects of the mitochondria-targeting antioxidant Mitoquinone (MitoQ) during cryopreservation of bull sperm. Semen was collected from 11 Simmental bulls (two ejaculates per bull) and diluted in Triladyl® supplemented with various concentrations of MitoQ (0, 0.2, 2, and 20 nM) to a final concentration of 65 × 106  sperm/ml. After thawing (0 and 3 hr), we assessed the following sperm traits: sperm motility by computer-assisted sperm analysis (CASA), DNA fragmentation index by SCSA® and plasma and acrosome membrane integrity, intracellular calcium concentration, esterase activity, mitochondrial membrane potential and synthesis of ROS using two multicolour flow cytometric assays. After 3 hr of incubation, 20 nM MitoQ increased (p < .05) sperm ROS synthesis compared to Control, whereas none of the other quality parameters were altered (p > .05). Therefore, we concluded that addition of MitoQ to semen extender before cryopreservation of bull sperm was unable to improve post-thaw sperm quality. Furthermore, 20 nM of MitoQ increased frozen-thawed sperm ROS synthesis, without apparent negative effects on the evaluated sperm traits.

Relationship between Na/K-ATPase in thawed sperm and fertility of Angus bulls.

Since bull fertility prediction remains challenging, the identification of potential fertility markers is important considering the economic benefits to the livestock industry. The main goal of this study was to determine the Na/K-ATPase activity and expression in thawed sperm of high (HF)- and low-fertility (LF) Angus bulls. Samples from three different batches/bulls with HF (n = 4) and LF (n = 4) were used. The Na/K-ATPase activity was determined after thawing, whereas sperm kinematics, membrane integrity, and expression of Na/K-ATPase on sperm surface were evaluated immediately post-thaw and after 120 minutes of incubation. Within the same incubation time, there was no difference on sperm membrane integrity, kinematics, and the expression of Na/K-ATPase on the sperm surface between HF and LF bulls. Kinematic parameters of LIN and VCL were not influenced by incubation time in samples from HF and LF, respectively. A tendency (P = 0.06) of higher Na/K-ATPase enzymatic activity for sperm of HF bulls compared to LF bulls was observed (0.49 ± 0.07 and 0.32 ± 0.06, respectively). In conclusion, Na/K-ATPase activity and expression in thawed sperm from Angus bulls are not related to the fertility index after fixed-time artificial insemination. However, sperm kinematics related to hyperactivation might indicate higher sperm cryotolerance for HF bulls.

Post-thaw quality of ram sperm frozen with different concentrations of low-density lipoproteins associated with non-enzymatic antioxidants.

The cryopreservation reduces ram sperm quality, decreasing the pregnancy rate of ewes inseminated with thawed sperm. Hence, we aimed to improve the post-thaw quality of ram sperm replacing egg yolk on Tris-Glucose extender with different concentrations of LDL (2 or 8%), associated with the addition of 10 mM non-enzymatic antioxidants (ascorbic acid, hydroxytoluene butylate, ascorbyl palmitate, and trehalose). Semen samples were collected from six rams, split into different treatments, and frozen. After thawing, kinematic (CASA), structural (propidium iodide and carboxyfluorescein diacetate) and functional (hypoosmotic test) sperm membrane integrity was assessed. Total motility, VCL, and LIN were also assessed in thawed samples during 3 h of incubation (38 °C). The results showed that hydroxytoluene butylate at 10 mM in Tris-Glucose extender with 8% LDL improved velocity parameters immediately post-thaw compared with Tris-Glucose egg yolk extender, as well as prevented the reduction of total motility and VCL after incubation. There was no benefit of adding ascorbic acid and trehalose. Moreover, for the first time, it was shown the motility impairment promoted by ascorbyl palmitate to ram sperm.

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts.

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.

Vincristine sulfate treatment influence on kidney function of female dogs with transmissible venereal tumor / Influência do tratamento com sulfato de vincristina na função renal de cadela com tumor transmissível venéreo

Chemotherapy agents have some undesirable and non-selective cytostatic effects. Considering that kidneys are vulnerable to drug-induced toxicity, this study evaluated renal injury caused by vincristine sulfate (VS) in 12 female dogs diagnosed with transmissible venereal tumor (TVT). The animals were treated with VS (0.025 mg/kg IV) every 7 days for 4 weeks. During treatment, the animals were subjected to clinical examination, blood count, serum measurement of symmetric dimethylarginine (SDMA), blood urea nitrogen (BUN), creatinine, alanine aminotransferase, and alkaline phosphatase. In addition, urinalysis and urinary gamma-glutamyl transferase (GGT) measurements were performed. All parameters were determined three times: before beginning the treatment (T0), after 14 days (T1), and after 28 days (T2). During the study period, there were no changes in serum urea or creatinine levels, urine specific gravity, or persistent proteinuria. Furthermore, urinary GGT measurement did not indicate tubular lesions, and consistent elevation of SDMA was found in only one patient above the reference range. The results showed that weekly therapy with VS as a single agent for 28 days does not induce renal injury in most cases.(AU)

Os agentes quimioterápicos possuem efeitos citostáticos indesejáveis e não seletivos. Considerando a vulnerabilidade renal à toxicidade induzida por drogas, este estudo avaliou a lesão renal causada pelo sulfato de vincristina (VS) em 12 cadelas com diagnóstico de tumor venéreo transmissível (TVT). Os animais foram tratados com VS (0,025 mg / kg IV) a cada sete dias, durante quatro semanas. No transcurso do tratamento, os animais foram submetidos a exame clínico, hemograma, dosagem sérica de dimetilarginina simétrica (SDMA), nitrogênio ureico sanguíneo (BUN), creatinina, alanina aminotransferase e fosfatase alcalina. Além disso, foram realizadas análises de urina e medições de gama-glutamil transferase (GGT) urinária. Todos os parâmetros foram mensurados em três tempos, antes do início do tratamento (T0), aos 14 dias (T1) e aos 28 dias (T2). Durante o período do estudo, não houve alterações nas concentrações de ureia ou creatinina séricas, na gravidade específica da urina ou proteinúria persistente. Além disso, a medição de GGT urinária não indicou lesões tubulares, e elevação consistente de SDMA foi encontrada em apenas um paciente acima do intervalo de referência. Os resultados mostraram que a terapia semanal com VS como agente único por 28 dias não induz lesão renal na maioria dos casos.(AU)

Distúrbios reprodutivos em cabras experimentalmente infectadas por Toxoplasma gondii / Reproductive disorders in does experimentally infected by Toxoplasma gondii

Objetivou-se descrever os distúrbios reprodutivos associados à infecção experimental por Toxoplasma gondii através da inseminação artificial com sêmen contaminado em quatro cabras no estágio crônico da infecção. As características do trato reprodutor foram avaliadas através de ultrassonografia transretal, visando o diagnóstico gestacional ou de desordens reprodutivas, após a infecção experimental. Ao final do experimento, os animais foram necropsiados e avaliações histopatológicas e PCR foram realizados. Dentre os animais infectados que exibiram mortalidade embrionária, duas apresentaram anestro e duas apresentaram repetição de estro, sendo que destas uma apresentou intervalos entre estros reduzido (sete dias) e outra em intervalo regular (21 dias). Todavia, ambas foram submetidas a monta natural durante os estros naturais subsequentes e não foi confirmada gestação até o final do experimento (90 dias). Duas cabras exibiram alterações nos exames de ultrassonografia, sendo identificadas um cisto ovariano, e uma hidrossalpinge, ambas confirmadas no exame post-mortem. As principais lesões microscópicas nesse grupo foram infiltração neutrofílica dos pulmões, glomerulonefrite intersticial e infiltração neutrofílica do fígado. O DNA de T. gondii foi encontrado nos órgãos (coração e cérebro) de três cabras. Em conclusão, cabras infectadas com sêmen contendo T. gondii no momento da inseminação artificial apresentam distúrbios reprodutivos na fase crônica da infecção que podem estar associados à toxoplasmose.

The aim of this study was to describe the reproductive disorders related to experimental infection by artificial insemination with semen contaminated with Toxoplasma gondii of four goats in the chronic phase of the infection. In the end of the study, the does were submitted to necropsy, and PCR and histopathological evaluations were performed. Among infected does that exhibited embryonic loss, two were in anestrus and two exhibited repeated estrus. One of the latter animals exhibited clinical signs of estrus at seven-day intervals, whereas the other had a 21-day estrous cycle. However, both does were naturally mated on subsequent natural estrous and were not able to get pregnant until the end of the experiment (90 d). Two of the goats exhibited abnormalities in the ultrasound examinations, one of which was an ovarian cyst, while the other was a hydrosalpinx, both of which were confirmed in the post-mortem examination. The main microscopic injuries in this group were neutrophilic infiltration of the lungs, interstitial glomerulonephritis and neutrophilic infiltration of the liver. T. gondii DNA was found in the organs (heart and brain) of three does. In conclusion, does infected with Toxoplasma gondii in semen at the time of artificial insemination display reproductive disorders in the chronic phase of infection that might be associated with toxoplasmosis.

Reproductive disorders in does experimentally infected by Toxoplasma gondii / Distúrbios reprodutivos em cabras experimentalmente infectadas por Toxoplasma gondii

The aim of this study was to describe the reproductive disorders related to experimental infection by artificial insemination with semen contaminated with Toxoplasma gondii of four goats in the chronic phase of the infection. In the end of the study, the does were submitted to necropsy, and PCR and histopathological evaluations were performed. Among infected does that exhibited embryonic loss, two were in anestrus and two exhibited repeated estrus. One of the latter animals exhibited clinical signs of estrus at seven-day intervals, whereas the other had a 21-day estrous cycle. However, both does were naturally mated on subsequent natural estrous and were not able to get pregnant until the end of the experiment (90 d). Two of the goats exhibited abnormalities in the ultrasound examinations, one of which was an ovarian cyst, while the other was a hydrosalpinx, both of which were confirmed in the post-mortem examination. The main microscopic injuries in this group were neutrophilic infiltration of the lungs, interstitial glomerulonephritis and neutrophilic infiltration of the liver. T. gondii DNA was found in the organs (heart and brain) of three does. In conclusion, does infected with Toxoplasma gondii in semen at the time of artificial insemination display reproductive disorders in the chronic phase of infection that might be associated with toxoplasmosis.

Objetivou-se descrever os distúrbios reprodutivos associados à infecção experimental por Toxoplasma gondii através da inseminação artificial com sêmen contaminado em quatro cabras no estágio crônico da infecção. As características do trato reprodutor foram avaliadas através de ultrassonografia transretal, visando o diagnóstico gestacional ou de desordens reprodutivas, após a infecção experimental. Ao final do experimento, os animais foram necropsiados e avaliações histopatológicas e PCR foram realizados. Dentre os animais infectados que exibiram mortalidade embrionária, duas apresentaram anestro e duas apresentaram repetição de estro, sendo que destas uma apresentou intervalos entre estros reduzido (sete dias) e outra em intervalo regular (21 dias). Todavia, ambas foram submetidas a monta natural durante os estros naturais subsequentese não foi confirmada gestação até o final do experimento (90 dias). Duas cabras exibiram alterações nos exames de ultrassonografia, sendo identificadas um cisto ovariano, e uma hidrossalpinge, ambas confirmadas no exame post-mortem. As principais lesões microscópicas nesse grupo foram infiltração neutrofílica dos pulmões, glomerulonefrite intersticial e infiltração neutrofílica do fígado. O DNA de T. gondii foi encontrado nos órgãos (coração e cérebro) de três cabras. Em conclusão, cabras infectadas comsêmen contendoT. gondii no momento da inseminação artificial apresentam distúrbios reprodutivos na fase crônica da infecção que podem estar associados à toxoplasmose.

Experimental vaginal infection of goats with semen contaminated with the "CPG" strain of Toxoplasma gondii.

The objective was to characterize the transmission of Toxoplasma gondii in goats experimentally infected vaginally with semen contaminated with the CPG strain (genotype III). Ten female goats were randomly allocated into 2 groups (G1 and G2), each with 5 animals, and inseminated during estrus. Goats in G1 were inseminated with semen containing 1 × 10(5) tachyzoites, whereas those in G2 (control) were inseminated with semen free from tachyzoites (insemination = day 0). In G1, seroconversion (indirect immunofluorescence reaction) and DNA (polymerase chain reaction) in the blood was present in 4/5 and 3/5, respectively, from the 7th day. In G2, all goats were negative in all tests. Embryonic reabsorption occurred in 4 of 5 goats from G1 between days 21 and 49. In conclusion, artificial vaginal insemination with semen containing tachyzoites of T. gondii -infected goats and is a potential transmission route of this parasite through semen.