Animal Reproduction, Volume: 18, Issue: 4, Published: 2021
  • Effect of pre-pubertal growth rate of Sohagi ram lambs on some physiological parameters and sexual behavioral patterns at puberty Original Article

    Elaref, Mohamed Youssef; Solouma, Gamal Mahmoud; Abdel-latif, Doaa Ahmed

    Abstract in English:

    Abstract Thirty healthy Sohagi ram lambs with an average age of 188.6±7.3 days were used to study the effect of pre-pubertal growth rate on some physiological parameters and sexual behavioral patterns at puberty. Ram lambs were divided into three groups (10 animals per each group) according to the previous growth rate until 6 months of age. Groups were marked as fast, medium and slow growing. Animal groups were housed in closed barns with access to an open area. Results showed that age and weight of ram lambs at puberty were significantly affected (P<0.05) by the pre-pubertal growth rate. Ram lambs in the fast growing group were reached to onset puberty firstly at 272.6 days with body weight (BW) 37.1 kg on average then ram lambs in medium group (284.8 days with BW 32.7 kg), while ram lambs in slow growing group were the last (314.1 days with BW 32.5 kg). Blood‎ testosterone‎ concentration at puberty was not significantly different among growing groups (1.494± 0.03 ng/ml on average, ranged from 1.287 to 1.902 ng/ml). Testes measurements from 6 months of age until puberty show that ram lambs in fast growing group had the highest values of testes length, circumference and volume followed by those in medium and slow growing group. Sexual behavioral observation showed that flehmen and mounting behavior were significantly higher for ram lambs in fast growing group (5.63 and 6.75 number/12h) than slow growing group (4.25 and 5.38 number/12h) while in medium growing group were intermediate (4.88 and 5.88 number/12h). From these findings, could be concluded that age, weight and sexual behavioral patterns of Sohagi ram lambs at puberty were affected by pre-pubertal growth rate, and the breeders should strive to achieve good growth rates for their lambs before puberty which led to improving reproductive performance.
  • Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats Original Article

    Fernandes, Denilsa Pires; Rossetto, Rafael; Montenegro, Assis Rubens; Fernandes, César Carneiro Linhares; Bravo, Pamela Angela; Moreno, Maria Eugenia; Cavalcanti, Camila Muniz; Kubota, Guilherme Araújo; Rondina, Davide

    Abstract in English:

    Abstract Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.
  • Ultrasonography of the nuchal translucency of healthy and abnormal English Bulldog fetuses Original Article

    Pavan, Letícia; Gasser, Beatriz; Maronezi, Marjury Cristina; Cruz, Igor Cezar Kniphoff da; Carneiro, Rafael Kretzer; Silva, Priscila; Ramirez Uscategui, Ricardo Andrés; Padilha-Nakaghi, Luciana Cristina; Feliciano, Marcus Antônio Rossi

    Abstract in English:

    Abstract The aim of this study was to measure the nuchal translucency (NT) of canine fetuses to establish reference values ​​for healthy gestational processes and to verify its effectiveness in the diagnosis of congenital abnormalities. On day 34 of gestation, the NT of three fetuses from each of the 26 English bulldog female dogs was measured. The first fetus was the one located immediately cranial to the bladder, the second was selected from the left side of the abdomen, and the third from the right side. The reference values for healthy animals were offset using descriptive statistics. The diagnostic ability of the test to identify fetal malformations was studied using receiver operating characteristic curve analysis. Of the 26 litters, only 18 had healthy fetuses, 4 had fetuses with anasarca, 3 had fetuses with abdominal wall defects, and 1 had both types of abnormalities. The NT was higher in canine fetuses that presented anasarca in the litter than in normal litters (1.8 ± 0.77 mm vs. 1.4 ± 0.48 mm; P = 0.0249), with a cut-off value of NT > 1.45 mm (sensitivity = 61.54%, specificity = 70.18%). NT greater than 1.45 mm seems to be a diagnostic tool for the identification of anasarca during gestation of bulldogs. Considering the unprecedented use of this parameter in canine species and the limitations found during the study, further studies will be needed in order to use it on clinical practice.
  • Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro Original Article

    Schefer, Letícia; Schwarz, Kátia Regina Lancelloti; Paschoal, Daniela Martins; Castro, Fernanda Cavallari de; Fernandes, Hugo; Botigelli, Ramon César; Leal, Cláudia Lima Verde

    Abstract in English:

    Abstract Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.
  • The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation Original Article

    Macêdo, Iara Nóbrega; Arruda, Lucia Cristina Pereira; Santana, Breno Barros de; Moura, Thalles Cloves Maciel de; Guerra, Maria Madalena Pessoa; Bezerra, Diogo Gutemberg; Carneiro, Gustavo Ferrer; Silva, Sildivane Valcácia

    Abstract in English:

    Abstract The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.
  • The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells Original Article

    Emamdoust, Fatemeh; Aminafshar, Mehdi; Zandi, Mohammad; Sanjabi, Mohammad Reza

    Abstract in English:

    Abstract The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old’s lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days’ culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.
  • Synchronization of follicular wave emergence does not improve embryonic yield in superovulated ewes Original Article

    Brasil, Oscar Oliveira; Moreira, Nathalia Hack; Conceição, Fábia Fernanda Cardoso de Barros da; Souto, Paula Lorena Grangeira; Silva, Cleidson Manoel Gomes da; Ramos, Alexandre Floriani

    Abstract in English:

    Abstract The present study aimed to investigate the effects of a combination of progesterone with different doses of E-17β on following end points: (1) ovarian follicular dynamics and emergence of a new follicular wave, and (2) superovulatory response and embryo yield. In Experiment 1, 28 ewes were randomly divided into four groups (n = 7) to receive either 2.0 mg, 1.0 mg, 0.5 mg or none E-17β one day after insertion of a progesterone device. The different doses of estradiol similarly delayed the moment of follicular emergence (overall mean = 3.1 ± 1.0 days vs. control group = 0.86 ± 1.0 days; P < 0.01), but the emergence of the new wave showed greater synchronization with the 0.5 mg dosage of E-17β. In Experiment 2, sixty-two donor ewes received an internal progesterone release device (day -1) for 7 d and 1 d after the insertion of this device (day 0) were allocated randomly to receive 0.5 mg of E-17β or only the vehicle (control group). Superstimulation was initiated on day 3 with the administration of 133 mg of pFSH in eight decreasing doses. Contrary to expectations, the protocol with the administration of 0.5 mg E-17β did not improve the percentage of donors with > 2 CL, the number of CL and the production of embryos (P > 0.05). It was concluded that the combination of progesterone and 0.5 mg E-17β was more efficient in synchronizing the emergence of the new follicular wave, however this approach seems to be unnecessary in ewe’s superovulation programs.
  • Indicators of immunosuppression peripartum in dual purpose cows in the tropics affected health, productive and reproductive parameters Original Article

    Lammoglia, Miguel Ángel; Avalos, Ivan; Cabrera, Amalia; Rojas, Maria Rebeca; Garcez, Nora; Tabarez, Abigail

    Abstract in English:

    Abstract The objective of the study was to identify immunosuppression peripartum indicators in dual purpose cows in the tropics and determine their effects on productive and reproductive parameters. The indicators used were: changes in leukocyte and neutrophils population, concentrations of energy metabolites (β-hydroxybutyrate and glucose) and body condition scores (BCS). Blood sampling and BCS (scale 1 – 5) were taken weekly during the peripartum. Uterine health was assessed (3 weeks postpartum) by ultrasonography and using a vaginal score (0-3) described by Sheldon et al. (2006). Cows (n=30) were classified as healthy or clinical endometritis (CE). CE prevalence was as high as 29.6%. Leukocyte and neutrophils populations diminished while in the peripartum and were lower (P<0.05) in cows suffering CE. Healthy cows had higher (P<0.05) daily milk production than those with CE (18.84±0.63 vs 14.76±0.84 kg). CE cows had lower (P<0.05) reproductive performance compared with healthy cows (open days: 244.40 ± 35.00 vs 178.00 ± 23.33 and services by conception 3.33 ± 0.51 vs 1.83 ± 0.34). BCS similarly (P>0.05) decreased following parturition in both groups. Concentrations of energy metabolites during peripartum fluctuated in a similar (P>0.05) manner in healthy and CE cows. In summary, dual purpose cows in tropical conditions, presented peripartum immunosuppression indicators, characterized by a decline in the leukocyte population, mainly neutrophils, as well as decreased glucose concentrations and BCS postpartum. In addition to it, there was a rise in the β-hydroxybutyrate concentrations and cows presenting CE had a negative effect in the productive and reproductive parameters.
  • Changes in ovarian morphology and hormone concentrations associated with reproductive seasonality in wild large Japanese field mice (Apodemus speciosus) Original Article

    Komatsu, Kazuki; Murata, Kohsuke; Iwasaki, Tsugumi; Tokita, Syun; Yonekura, Shiina; Sugimura, Satoshi; Fujishima, Yohei; Nakata, Akifumi; Miura, Tomisato; Yamashiro, Hideaki

    Abstract in English:

    Abstract Wild large Japanese field mice (Apodemus speciosus) responses to cyclic seasonal changes are associated with physiological and behavioral changes. However, the detailed regulation of oogenesis in the ovary during the seasonal reproductive cycle in wild large Japanese field mice has not been studied. We assessed the dynamics and changes in ovarian morphology and hormone concentrations associated with reproductive seasonality throughout the year. The stages of the ovarian morphological breeding cycle of wild large Japanese field mice were classified as breeding, transition, and non-breeding periods during the annual reproductive cycle. Measurement of blood estradiol concentrations throughout the year showed that the levels in September and October were higher than those in other months. It is presumed that follicle development starts from a blood estradiol concentration of 38.4 ± 27.1 pg/mL, which marks a shift from the transitional season to the breeding season, followed by the transition to the non-breeding season at 26.1 ± 11.6 pg/mL. These results suggest that seasonal follicle development in wild rodents is correlated with estradiol regulation. We consider this species to be an alternative animal model for studying seasonal reproductive changes and the effects of environmental changes.
  • The neuroendocrine pathways and mechanisms for the control of the reproduction in female pigs Review Article

    Zhao, Shuang; Guo, Zongyi; Xiang, Wei; Wang, Pingqing

    Abstract in English:

    Abstract Within the hypothalamic-pituitary-gonad (HPG) axis, the major hierarchical component is gonadotropin-releasing hormone (GnRH) neurons, which directly or indirectly receive regulatory inputs from a wide array of regulatory signals and pathways, involving numerous circulating hormones, neuropeptides, and neurotransmitters, and which operate as a final output for the brain control of reproduction. In recent years, there has been an increasing interest in neuropeptides that have the potential to stimulate or inhibit GnRH in the hypothalamus of pigs. Among them, Kisspeptin is a key component in the precise regulation of GnRH neuron secretion activity. Besides, other neuropeptides, including neurokinin B (NKB), neuromedin B (NMB), neuromedin S (NMS), α-melanocyte-stimulating hormone (α-MSH), Phoenixin (PNX), show potential for having a stimulating effect on GnRH neurons. On the contrary, RFamide-related peptide-3 (RFRP-3), endogenous opioid peptides (EOP), neuropeptide Y (NPY), and Galanin (GAL) may play an inhibitory role in the regulation of porcine reproductive nerves and may directly or indirectly regulate GnRH neurons. By combining data from suitable model species and pigs, we aim to provide a comprehensive summary of our current understanding of the neuropeptides acting on GnRH neurons, with a particular focus on their central regulatory pathways and underlying molecular basis.
  • Cell differentiation events in pre-implantation mouse and bovine embryos Review Article

    Carreiro, Letícia Escobar; Santos, Gabriel Siqueira dos; Luedke, Felipe Eduardo; Goissis, Marcelo Demarchi

    Abstract in English:

    Abstract Early mammal embryogenesis starts with oocyte fertilization, giving rise to the zygote. The events that the newly formed zygote surpasses are crucial to the embryo developmental success. Shortly after activation of its genome, cells of the embryo segregate into the inner cell mass (ICM) or the trophectoderm (TE). The first will give rise to the embryo while the latter will become the placenta. This first segregation involves cellular and molecular processes that include cell polarity linked to intracellular pathway activation, which will regulate the transcription of trophectoderm-related genes. Then, cells of the ICM undergo the second event of mammalian cell differentiation, which consists of the separation between epiblast (EPI) and hypoblast or primitive endoderm (PrE). This second segregation involves paracrine signaling, leading to differential expression of key genes that will dictate the fate of the cell. Although these processes are described in detail in the mouse, recent studies suggest that the bovine embryo could also be an interesting model for early development, since there are differences to the mouse and similarities with early human embryogenesis. In this review, we gathered the main data available in the literature upon bovine and mouse early development events, suggesting that both models should be analyzed and studied in a complementary way, to better model early events occurring in human development.
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