Effects of the supplementation with an high‐polyphenols extra‐virgin olive oil on kinetic sperm features and seminal plasma oxidative status in healthy dogs

The aim of this work was to evaluate the effects of the supplementation of two extra‐virgin olive oils (EVOO) having different polyphenols content, on canine spermatozoa kinetic parameters and seminal plasma oxidative status. The study was conducted on 12 clinically healthy dogs of different breeds (2–7 years, 5–48 kg of body weight) divided into two groups: an experimental group supplemented with EVOO (Coratina cultivar) high in polyphenols (H‐P) and a control group fed EVOO (Cima di Bitonto cultivar) low in polyphenols (L‐P). The oil was daily administered per os (1 ml/3 kg BW) before meal. Semen collection was made twice at 15 days distance (D0₁ and D0₂) and then at 30 (D30), 60 (D60) and 90 (D90) days. Semen concentration and kinetic parameters were measured using computer‐assisted sperm analysis (CASA) system to evaluate: sperm total count, sperm motile (MOT%), progressive motility (PROGR%) and its fractions, straight‐line velocity (VSL, μm/s), curvilinear velocity (VCL, μm/s), average path velocity (VAP, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz), straightness (STR%) and linearity (LIN%). On seminal plasma, reactive oxygen species (ROS) and biological antioxidant potential (BAP) were tested. From findings, no differences were found for sperm MOT, VSL, VCL, VAP, ALH, BCF, STR, LIN and BAP. A gradual enhancement of PROGR% was observed in H‐P group (p < .01). The ROS levels were higher in dogs H‐P compared to the other group (p < .05). In conclusion, our results highlight the positive effects of EVOO polyphenols on sperm PROGR% in healthy dogs.     

Effect of body condition score and reuse of progesterone‐releasing intravaginal devices on conception rate following timed artificial insemination in Nelore cows

This study had the aim of investigating the efficiency of timed artificial insemination (TAI) through the progesterone‐releasing intravaginal device (PRID), used in new condition and for the second and third times in Nelore cows. The effects of device reuse and body condition score (BCS) on the conception rate (CR) were evaluated in 1,122 multiparous Nelore cows (mean BCS of 2.7 ± 0.4), which were randomly distributed into three groups that received new (n = 330), once (n = 439) and twice used (n = 353) PRID. Among the 1,122 females that underwent TAI, 573 became pregnant, thus representing an overall CR of 51.06%. Cows with BCS between 2.75 and 4.0 had greater (p < .0001) CR (69.75%) than cows with BSC of 2.0–2.5 (32.98%). It was observed that the CR through using PRID was 60.00%, 51.71% and 41.93% for new, once and twice used PRID, respectively, with difference between all groups (p < .0001). Under tropical conditions, animals with BCS greater than 2.5 had a higher CR, and the CR decreased proportionally with the number of times that the PRID had been used.     

Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes

In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48 h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.     

Midgut cysteine-proteinase activity in the velvetbean caterpillar (<Emphasis Type="Italic">Anticarsia gemmatalis</Emphasis> (Hübner))

Proteinase inhibitors are currently targeted as potential insect control agents, but adaptation to proteinase inhibitors is a recognized limitation to such approach requiring the understanding of how phytophagous species can cope with such compounds. The velvetbean caterpillar (Anticarsia gemmatalis) is a key pest of soybean and well-adapted to its host proteinase inhibitors, which is rich in serine-proteinase inhibitors, particularly trypsin-like proteinase inhibitors. As the expression of cysteine proteinases in the midgut of the velvetbean caterpillar is a potential adaptation to circumvent its host defense, we assessed and characterized the digestive cysteine-proteinase activity from velvetbean caterpillars. Significant soluble and membrane-bound proteolytic activity was obtained and was consistent with those of cysteine proteinases based on the substrate and inhibitors used for their characterization. The K _m and V _max values obtained were 2.35 ± 0.50 mM and 40.89 ± 6.68 nmol min⁻¹ mg⁻¹ for the soluble proteinases and 0.33 ± 0.03 mM and 24.54 ± 0.67 nmol min⁻¹ mg⁻¹ for the membrane-bound proteinases, range of values also consistent with cysteine proteinases. Therefore, the proteolytic activity observed in the velvetbean caterpillar midgut is consistent with that of cysteine proteinases providing preliminary support for the contention of their potential involvement mitigating the negative effects of serine-protease inhibitors in this species.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Expression of the μ Opioid Receptor and Effects of the Opioid Antagonist Naloxone on In Vitro Maturation of Oocytes Recovered from Anoestrous Bitches

The μ-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus–oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 × 10⁻⁶, 1 × 10⁻⁸ and 1 × 10⁻¹⁰  m . The rate of oocytes resuming meiosis after culture in presence of 1 × 10⁻⁶  m Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as δ and κ, and possible interactive effects of their antagonists on canine oocyte maturation.     

Transfer of Bovine Blastocysts Derived from Short-term In Vitro Culture of Low Quality Morulae Produced In Vivo

The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus® or TCM‐199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5°C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non‐cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor‐recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non‐cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non‐cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short‐term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients.