Abstract in English:

Abstract Due to their self-renewal and differentiation potentials, mesenchymal stem cells (MSCs) may be induced into germ cells (GC) differentiation under in vitro conditions. In veterinary medicine, this technology could provide an alternative method to artificial insemination, as well as potentially useful for the conservation of endangered species. Previous studies have reported the use of SCs and MSCs co-culture systems, as well as SCs conditioned medium (SCCM) to induce GC differentiation of human and murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The objective of this study was to evaluate the effect of SCCM as an inducer of in vitro GC differentiation of MSCs derived from fetal bovine adipose tissue (AT-MSCs). SCCM was collected from bovine SC cultures generated from adult bull testis. The effect of SCCM on MSCs was analyzed using quantitative PCR (Q-PCR) and flow cytometry. CD73 mRNA levels were decreased (P<0.05) in AT-MSC/SCCM at day 14 of culture compared to control. CD90 and CD105 gene expression were detected during the 21 days of culture; however, relative expression levels were not different (P>0.05) between treated and controls cells. DAZL gene expression was detected on day 21 of culture, as well as a proportion of AT-MSC positive for DAZL at day 21 of culture. OCT4, PIWIL2 and DAZL gene expressions were detected from day 0, 7 and 21 of culture, respectively, as well as a proportion of cells positive for each marker were detected at day 21 of culture. However, similar gene and protein expression levels (P>0,05) were detected between AT-MSCs/SCCM and control cultures. DMC1 gene expression levels were detected from day 7 of culture, and expression levels were not different (P>0,05) between treatment and control cells. Expression patterns of MSC, pluripotent, GC and meiotic markers indicate that SCCM did not induce GC differentiation of AT-MSCs.

Abstract in English:

Abstract The objective of this study was to evaluate the effect of adding Sicilian lemon and wild orange essential oil nanoemulsion, using soy lecithin as a surfactant, to ram semen freezing extender. The nanoemulsions were prepared by high-energy emulsification method using soy lecithin (5%) as a surfactant. The organoleptic and physicochemical characteristics were evaluated. Semen samples (n = 7) obtained from adult rams (n = 6) were frozen in a Tris-egg yolk extender supplemented with Sicilian lemon or wild orange nanoemulsion at different concentrations (0.0%, 1.5%, 2.5%, and 3.5%). After thawing (37oC, 30 s), the samples were evaluated for kinematics, plasma and acrosomal membrane integrity, and mitochondrial membrane potential. Visually, the nanoemulsions of Sicilian lemon or wild orange essential oil appeared homogeneous, fluid, opaque, without lumps, odorless, and colored, immediately after preparation (0 h) and after thermal stress (24 h). The physicochemical characterization of the nanoemulsions showed vesicles with average sizes < 220.00 nm, polydispersity index < 0.30, and zeta potential of -59.00 mV. Semen samples from the groups treated with Sicilian lemon (1.5%, 2.5%, and 3.5%) or wild orange (1.5%, 2.5%, and 3.5%) nanoemulsions did not differ (P ≤ 0.05) in terms of kinematics, plasma membrane integrity, and mitochondrial membrane potential when compared to the control group. However, the groups treated with Sicilian lemon (2.5% and 3.5%) and wild orange (1.5%, 2.5%, and 3.5%) nanoemulsions had a higher percentage (P ≤ 0.05) of cells with intact acrosomes when compared to the control group. It can be concluded that nanoemulsions of essential oils of Sicilian lemon (2.5% and 3.5%) and wild orange (1.5%, 2.5%, and 3.5%), using soy lecithin (5%) as a surfactant, can be used as additives to the Tris-egg yolk extender for ram semen freezing due to their ability to preserve the acrosome post-thawing.

Abstract in English:

Abstract Leptospirosis is a zoonosis caused by bacteria of the genus Leptospira. In cattle, the infection mainly manifests in the genital form. However, there are still few studies about this manifestation. The aim of this study was to describe the control of an outbreak of leptospirosis in dairy cows with reproductive disorders, through the combination of diagnostic methods and the integration of vaccination with antibiotic therapy. Between December 2022 and April 2023, 17 cows presented reproductive disorders. After the outbreak, two consecutive blood collections and one cervicovaginal mucus (CVM) collection were carried out. The blood samples were tested by the microscopic seroagglutination test (MAT), using a collection of antigens with eight strains of Leptospira and a cutoff point ≥ 1:200. The CVM samples were analyzed by polymerase chain reaction (PCR), with the lipL32 gene as target. The control was carried out with the CattleMaster® GOLD FP 5/L5 vaccine, in addition to the application of streptomycin (25 mg/kg) in positive cows. After one year of sanitary management, the CVM PCR was repeated to evaluate the effectiveness of the integrated control. In serology, 58.8% (10/17) of the cows were reactive, with 100% (10/10) for the serogroup Sejroe. In the molecular analysis, 58.8% (10/17) of the cows were positive. When combining the two methods, the result was 82.3% (14/17) reagent/positive. After the integrated control, 0.0% (0/17) of cows were positive. It was concluded that the outbreak was related to bovine leptospirosis. Furthermore, the combination of diagnostic methods and integrated control proved to be efficient.

Abstract in English:

Abstract A total of 1600 juvenile Astyanax lacustris (commonly known as yellowtail lambari) with an initial age of two months were used. Fish were subjected to two systems: biofloc technology (BFT) and clear water recirculation (RAS) in a completely randomized design. Replicates were established for each treatment, and carbon sources and carbon ratios were adjusted specifically for BFT tanks to optimize microbial floc formation. Feeding was based on 3% of the total biomass of each tank, which was reduced to 1% when the fish reached four months of age. The gonadal factor and gonadosomatic index (IGS) were superior in fish cultured in the RAS system during the third month of culture, although all gonads from both BFT and RAS systems showed reproductive capability based on histological analysis. The hepatosomatic index (IHS) was higher in the BFT system in the third month. BFT males exhibited a higher percentage of dry matter and ether extract in body composition, while RAS males had a higher percentage of crude protein and ash. At five months, RAS males displayed superior total progressive motility, rapid sperm count, and flagellar beat frequency compared to BFT males. By fourteen months, RAS males had sperm with higher total motility, VSL (curvilinear velocity), VSL (linear velocity), and VAP (average trajectory velocity) than BFT males. Based on these results, BFT proves effective for the general cultivation and reproductive maintenance of Astyanax lacustris, although RAS offers slight advantages in seminal quality for male fish.

Abstract in English:

Abstract Visfatin is an adipokine involved in the regulation of female reproduction. However, there are no studies on the effects of visfatin on the in vitro culture of ovarian tissue in any species. Therefore, the aims of this study were to analyze the effects of visfatin on survival, primordial follicle activation, granulosa cell proliferation, and the immunostaining of tumour necrosis factor-α (TNF-α) in preantral follicles after the in vitro culture of sheep ovarian tissue. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or in α-MEM+ supplemented with different concentrations of visfatin (1 or 10 ng/mL) for 7 days. Subsequently, ovarian tissue was destined to histology (morphology, activation and growth) and immunohistochemistry (granulosa cell proliferation and pro-inflammatory cytokine TNF-α immunostaining). The results indicated that treatments with visfatin (1 or 10 ng/mL) maintained the percentage of morphologically normal follicles at a level similar (P>0.05) to the fresh control and significantly higher than of α-MEM+. A significant increase in primordial follicle activation was also observed in tissue cultured for 7 days at both visfatin concentrations compared to the fresh control and α-MEM+. In addition, only the treatment containing 10 ng/mL of visfatin significantly increased follicular and oocyte diameters, and granulosa cell proliferation compared to α-MEM+, and attenuated inflammatory activity by reducing TNF-α immunostaining after in vitro culture. In conclusion, 10 ng/mL visfatin maintains survival, reduces immunostainig of TNF-α and promotes the activation of primordial follicles by stimulating granulosa cell proliferation after the in vitro culture of sheep ovarian tissue.

Abstract in English:

Abstract This study aimed to generate yak-specific polyclonal antibodies against transforming growth factor beta 2 (TGF-β2). Specific primers targeting the TGF-β2 coding sequence (CDS) were designed, and the gene was amplified via RT-PCR. The amplified product was cloned into the pET-32a(+) vector to construct the recombinant plasmid pET-32a(+)-TGF-β2. This plasmid was transformed into Escherichia coli BL21(DE3) for protein expression. Isopropyl β-D-1-thiogalactopyranoside (IPTG) induced TGF-β2 production, and the recombinant protein was purified. New Zealand rabbits were immunized with the purified protein to generate polyclonal antibodies. Polyclonal antibody titers were determined using ELISA, while specificity was assessed through Western blot and immunohistochemistry. The recombinant plasmid was successfully constructed, and IPTG induction yielded a 63 kDa protein. Optimal expression occurred at 25 °C with 0.5 mmol·L−1 IPTG and a 10-hour induction period. ELISA confirmed an antibody titer of 1:106. Western blot and immunohistochemistry demonstrated TGF-β2 expression in female yak ovaries, oviducts, and uteri across reproductive stages, with significantly elevated ovarian levels during pregnancy. This study successfully produced and validated a highly specific anti-yak TGF-β2 polyclonal antibody, providing a vital tool for investigating its role in yak reproductive physiology.

Abstract in English:

Abstract This study aimed to characterize the ovarian follicular dynamics in locally adapted Curraleiro Pé-Duro cows and heifers. Cyclic heifers (n =12) and non-lactating, multiparous cows (n = 11) were examined daily by ultrasonography for two consecutive ovulations (an estrous cycle). Follicles > 3 mm and corpus luteum (CL) were measured and followed until they disappeared. Follicular and luteal characteristics were not different between heifers and cows. Consequently, data on cows and heifers were combined according to the number of follicular waves. Follicular dynamics was characterized by the predominance of two (36.8%) and three (63.2%) follicular waves. No difference in estrous cycle length between these follicular wave patterns was observed. The number of recruited follicles was smaller in the second follicular wave. The ovulatory follicle (OF) growth rate (mm/d) and maximum diameter were greater (P < 0.05) in females, showing three waves. The ovulatory wave was shorter (P < 0.05) than the preceding waves regardless of the wave pattern. No difference was found in CL development between females with two and three follicular wave patterns. Some follicular dynamics characteristics were similar to Bos taurus and others similar to Bos indicus, confirming the crosses made throughout the years. The data from this study will be useful to better estrous cycle manipulation aiming for good results in artificial insemination (AI), fixed-time AI (FTAI), and multiple ovulation and embryo transfer (MOET) programs.

Abstract in English:

Abstract To establish a mouse embryo bank at the Institute of Science and Technology in Biomodels, Oswaldo Cruz Foundation (ICTB/FIOCRUZ), embryos from genetically modified strains were vitrified. The strains included B6.129SVEV-CCBP2 (D6), B6.129P2-Nos2 (Nos2), B6.129S2-Cd28 (Cd28), B6.129P2-Ccl3 (Ccl3), B6.129S2-Alox5 (Alox5), B6.129P2-Ccr2 (Ccr2), B6.129P2-Ccr5 (Ccr5) and B6.129S1-Tlr6 (Tlr6). To accomplish this, the animals were superovulated and mated, and their embryos were collected and vitrified. The success of the technique was evaluated by examining the development of the embryos through thawing and in vitro culture, comparing them to a control group. The results were analyzed using percentages, Tukey's t-test, and Analysis of Variance. The embryonic development percentages for the different strains were as follows: D6 (55%), Nos2 (24.7%), Cd28 (45.8%), Ccl3 (50%), Alox5 (4.8%), Ccr2 (66.7%), Ccr5 (63.04%) and Tlr6 (52.8%). Significant differences were observed between the strains Nos2 (p=0.0434), Cd28 (p=0.034), Ccl3 (p=0.0006), and Alox5 (p=0.0166) compared to their respective control groups. In conclusion, the strains Ccr2 (p= p=0.0889), Ccr5 (p=0.0806), D6(p=0,0685) and Tlr6 (p=0.0806) demonstrated favorable results in terms of the vitrification protocol and subsequent embryonic development, as they did not significantly differ from the control groups.

Abstract in English:

Abstract The aim of this study was to evaluate the effect of follicular wave synchronization and equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) in Braford cows on the oocyte competence, maturation rate, and in vitro embryo production. Cows (n = 27) were divided into three groups on a crossover model: no treatment prior to OPU (Control), follicular wave synchronization (Synchro), and synchronization plus 800IU of eCG (eCG800). Donors of the groups Synchro and eCG800 were synchronized with 2 mg of estradiol benzoate (EB), prostaglandin F2α analogue (PGF2α) and intravaginal device with 1g of progesterone (P4) on D0. On day 3, eCG800 group donors received 800IU of eCG. On day 6, OPU was performed, and the number of follicles were counted and classified by diameter in small, medium, and large. In experiment 1, the viable oocytes were evaluated for competence development, nuclear maturation, and mitochondrial reorganization. In experiment 2, oocytes were matured, fertilized, and cultured in vitro to blastocyst stage. All analysis was performed by ANOVA, and the differences were compared by Tukey's test with significance P ≤ 0.05. The use of 800 IU of eCG increased (P < 0.05) the number of medium and large follicles compared to the Syncro group. The oocyte recovery, viability, nuclear or cytoplasmic maturation, cleavage, and grade 1 embryos rate did not differ among groups (P > 0.05). The blastocyst rate on D7 showed tendency (P = 0.075) to improve from Control (17±6.08%) to Synchro (23.8±8.95%) to eCG800 (37.3±6.51%). The dose of 800 IU of eCG 72 h before OPU increased the proportion and number of medium and large follicles in relation to the Control and Synchro groups, without affecting oocyte competence and tending to produce more blastocysts on D7.

Abstract in English:

Abstract The genes identification involved in male reproduction and the evaluation of its functions improve the comprehension about spermatogenesis molecular bases, fertilization, embryos early cleavage, spermatic quality and male infertility. The present study aimed to verify the Protamine1 (PRM1), Protamine2 (PRM2) and Cation Channel Sperm Associated 1 (Catsper1) genes expression into the equine sperm and their relations with the stallions' spermatic quality and fertility. Semen collections were performed in eighteen stallions, which were divided in two groups, based on fertility rates: fertile (with pregnancy rate per cycle ≥ 70%) and subfertile (with pregnancy rate per cycle ≤ 40%). The semen analysis was performed by Computer Assisted Sperm Analysis AndroVision®. The mRNA was extracted from the spermatozoa and the PRM1, PRM2 and Catsper1 gene expression verification in the spermatic cell was conducted by the qPCR technique. The results present a higher expression of PRM1 and Catsper1 in the fertile stallions’ group than subfertile group; there was no correlation of PRM1 and PRM2 expression with spermatic quality parameters; there was correlation of the Catsper1 expression with morphology and motility parameters. Negative correlation was found between the PRM1/PRM2 ratio, fertility and motility parameters. The present research demonstrates that the PRM1 and Catsper1 genes are related to stallions’ fertility and spermatic quality, and they may work as biomarkers.

Abstract in English:

Abstract The aim of this study was to evaluate the effects of supplementing the cryodiluent medium with sulfated polysaccharides (SP) extracted from marine algae (Ascophyllum nodosum or Solieria filiformis) and fish skin (Colossoma macropomum, Prochilodus brevis, or Oreochromis niloticus) on the cryopreservation of tambaqui semen. Twenty male tambaqui were used for semen collection and cryopreservation. For the fertilization assay, three males and five females were used. In Experiment 1, different concentrations of SP (0.0, 0.1, 0.25, 0.5, and 0.75 mg/mL) extracted from fish skin or marine algae were added to the freezing medium for C. macropomum semen. In Experiment 2, the results of sperm velocity analyses were used to select one concentration of each sulfated polysaccharide for use in fertilization trials. Among the treatments, A. nodosum at 0.75 mg/mL and C. macropomum at 0.50 mg/mL stood out, significantly improving sperm parameters such as motility, VCL, VSL, VAP, and LIN compared to the control group. S. filiformis, P. brevis, and O. niloticus also showed good results, with performance varying by concentration. Membrane integrity was higher in the algae-derived extract groups. Sperm morphology and DNA integrity did not differ significantly among groups. Fertilization rates remained high across all treatments (84.67% to 88.67%), with no statistically significant differences, indicating that the tested extracts did not compromise fertility. It was concluded that supplementation with SP from A. nodosum at 0.75 mg/mL and C. macropomum at 0.50 mg/mL, although all treatments showed similar fertility rates, is recommended as an additive to the semen dilution medium for tambaqui during freezing, as it improved important sperm parameters such as motility and VCL.

Abstract in English:

Abstract The application of assisted reproductive techniques (ART) in both farm animals and humans has faced challenges since its inception. Advances in this field have largely depended on a deeper understanding of the metabolic requirements and molecular dynamics of sperm, oocytes, and embryonic development. Glucose, for instance, is commonly utilized as an energy source in in vitro procedures. The pentose phosphate pathway (PPP), a pathway parallel to glycolysis, plays a key role in redox regulation via NADPH generation and ribose biosynthesis. This review highlights the role of the PPP in reproductive cells and discusses its potential implications for ART procedures.

Abstract in English:

Abstract Application of assisted reproduction techniques are essential for the preservation of endangered species, and ultrasonography has emerged as an interesting tool in this process, allowing noninvasive assessment of reproductive stages and characterization of male gonads. This review provides a compilation on the applications and perspectives of using ultrasonography for investigation of the morphological and functional aspects of the male reproductive tract in wild species. The technique, which has been improved with the use of vascular doppler, allows detailed analysis of blood flow and aids in the selection of individuals for breeding programs. Although there are challenges, such as physiological variations among species and the difficulty for applying ultrasonography to birds and reptiles, advances in imaging technologies, including elastography and doppler, have expanded the possibilities for diagnosis and monitoring reproductive status in various mammals. Ultrasonographic analysis contributes to the assessment of fertility, detection of testicular diseases and the definition of protocols for reproductive management, becoming an important tool in the conservation of wildlife and in the development of more effective assisted reproductive technologies.

Abstract in English:

Abstract Efficient spermatogenesis in mammals occurs when testicular temperature is approximately 2 to 8 °C below body temperature. Elevated testicular temperature can trigger oxidative stress and compromise sperm integrity during spermatogenesis, potentially resulting in damaged spermatozoa and male infertility. This study aimed to evaluate how heat stress affects the quantity of seminiferous tubules, and the abundance of germ cells within the seminiferous tubules. To this end, six Santa Inês rams were subjected to testicular insulation for 12 consecutive days, followed by two hemi-orchiectomies, the first 24 hours after insulation period to evaluate the immediate effect, and the second 30 days after the first hemi-orchiectomy to evaluate the late effect. Six Santa Inês rams composed the control group. Histological analyses were conducted to quantify the number of seminiferous tubules and the types of cells within them (spermatogonia, spermatocytes, and spermatids) in testicular fragments. Despite an increase in testicular temperature, no significant differences were observed in the number of seminiferous tubules. These findings probably reflect the resistance of Santa Ines rams to high environment temperatures. Regarding the abundance of cells, a decrease in spermatogonia (0.27% ± 0.06; 0.05% ± 0.03, p = 0.005) and an increase in spermatocytes (35.90% ± 1.58; 46.77% ± 4.33, p = 0.028) were observed immediately after the insulation period compared to 30 days after, the late effect. This result suggests an effect of the first hemi-orchiectomy on the remaining testicle, probably an attempt to maintain sperm production.

Abstract in English:

Abstract We tested the effects of centrifuging in vitro matured bovine oocytes for varying times on embryo development and cryotolerance. The oocytes were divided into four groups: control (GC) and centrifuged groups [5433 x g: G5, n = 463 (5 min); G10, n = 461 (10 min); and G15, n = 483 (15 min)]. After centrifugation, the oocytes underwent in vitro fertilization for embryo production. Two parameters were evaluated: i) embryonic development (n = 1,878), and ii) cryotolerance evaluation (survival and hatching rates; n = 303). The CG and G10 groups showed blastocyst rates of 42.25% and 45.77%, respectively, higher than those of the other groups (p = 0.02). The hatching rate was equal (p > 0.05) in CG (91.96%), G5: (87.74%), and G10: (95.73%) groups; however, it was lower in G15: 77.06% (p < 0.01). In the CG group, 65.88% of cryopreserved embryos survived, which was different (p < 0.05) from that in G5 (82.02%) and G10 (82.28%) (p > 0.05). Post-freeze hatching percentage was 74.0%, 87.7%, and 47.7%, in G5, G10, and G15, respectively, which was significantly greater than that in CG (p < 0.01; 26.8%). Post-freeze hatching percentage in only G10 matched that of the non-cryopreserved embryos CG (p = 0.06, 92%). We conclude that oocyte centrifugation for 10 minutes was efficient for in vitro embryonic development and cryopreservation of cattle embryos.