Flow cytometric evaluation of canine follicular cell apoptosis during the oestrous cycle.

Apoptosis is the cellular mechanism of ovarian follicular atresia in mammals; the aim of this study was to examine the apoptosis-related cyclic changes in follicular cells of different-sized antral follicles throughout the oestrous cycle in canines. Ovaries were collected from 26 adult female dogs (1-4 years) following routine ovariohysterectomy. Antral follicles were classified as small, medium or large antral or preovulatory. The percentage of apoptotic cells was determined flow cytometrically using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) DNA nick end labelling (TUNEL) assay. Apoptosis rate was quantified as the percentage of TUNEL-positive cells on a logarithmic scale. Percentages of TUNEL-positive cells obtained in the flow cytometric assay were compared among oestrous phases and follicular sizes using analysis of variance. Apoptotic follicles were observed in all types of canine follicles in different cycle phases and stages of development, possibly corresponding to the physiological process of the oestrous cycle. Both the oestrous phase and follicular size significantly influenced the apoptosis rate (p < .05). Apoptosis rate increased significantly (p < .05) as follicular development progressed. Apoptosis rate was the highest in large follicles during the oestrous phase (9.2%; p < .05) and the lowest in small follicles during the anestrus period (1.8%; p < .05). In conclusion, our results demonstrate significant differences in the apoptosis rate during the oestrous cycle related to follicle development in the canine ovary. Furthermore, flow cytometry using the TUNEL assay was found to be an effective method for detecting apoptosis in canine follicles.

Gene and protein expression of connexins 37 and 43 in cumulus-oocytes complexes throughout the canine oestrous cycle.

The aim of this study was to evaluate the expression of connexin (Cx) 37 and Cx43 in canine cumulus-oocyte complexes (COCs) during the oestrous cycle. Cx localisation was analysed by immunohistochemistry and immunofluorescence, whereas protein and gene expression was evaluated by western blotting and quantitative polymerase chain reaction respectively; comparisons were made using analysis of variance. Both Cx37 and Cx43 were expressed in all follicular stages; Cx43 was identified in cumulus cells and Cx37 was identified in cumulus cells, zonae pellucida and oocytes. Immunofluorescence analyses showed that Cx37 remained unchanged during the preovulatory stage but decreased after ovulation, whereas Cx43 remained unchanged before and after ovulation. Cx43 transcripts increased (P<0.05) during anoestrus and dioestrus in medium-sized follicles but remained unaltered during the pro-oestrus and antral stages during oestrus, before and after ovulation. Cx37 mRNA levels decreased in ovulated COCs (P<0.05). The highest levels of Cx37 protein (P<0.05) were detected in the preantral stage during anoestrus. In contrast, strong Cx43 signals were detected in oestrus and in medium-sized antral follicles in dioestrus (P<0.05). Overall, we demonstrated that Cx37 and Cx43 exhibit different expression patterns, suggesting specific roles throughout growth. Maintenance of Cx expression before ovulation indicates the involvement of Cx37 and Cx43 in the prolonged meiotic arrest.

Influence of growth differentiation factor 9 and bone morphogenetic protein 15 on in vitro maturation of canine oocytes.

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72 hr in a supplemented TCM199-Hepes medium as (a) Control group; (b) GDF-9 antibody (Ab); (c) BMP-15 Ab; (d) recombinant human (rh) GDF-9; (e) rh BMP-15 or (f) rh BMP-15 and GDF-9. Data were evaluated by ANOVA. The Abs against GDF-9 or BMP-15 had a negative impact on meiotic development. Higher (p < 0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF-9 Ab (64.4 ± 2.1%) or BMP-15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4 ± 7.8%). In contrast, more (p < 0.05) oocytes in control group reached MI (37.4 ± 1.3%) and MII stages (10.2 ± 2.1%) comparing to those groups with GDF-9 Ab (23.1 ± 4.7% MI; 0.0% MII) or BMP-15 Ab (16.4 ± 2.4%MI; 5.9% ± 2.1 MII). Higher rates (p < 0.05) of oocytes in control group stayed still arrested at GV (19.9 ± 8.6%) in comparison to those cultured with either rhGDF-9 (3.7 ± 0.4%) or rhBMP-15 (10.9 ± 0.7%). However, there were no differences in MII rates between oocytes cultured with GDF-9 (14.7 ± 3.1) and BMP-15 (7.8 ± 2.5) separately. But, more oocytes (p < 0.05) reached the MII stage (20.5 ± 3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs.

Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses. RESULTS: Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes. CONCLUSION: The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine.

Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids.

Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 × 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.

Dynamic Expression of Follicle-Stimulating Hormone and Estrogen mRNA Receptors Associated with microRNAs 34a and -let-7c in Canine Follicles during the Estrous Cycle.

The genes encoding for estrogen receptor (ESR2) and follicle-stimulating hormone receptor (FSHR) play crucial roles in ovarian follicular development. This study aimed to determine the expression levels of miRNAs predicted against FSHR and ESR2 mRNAs in follicular cells related to their target genes during the estrous cycle in canines. Antral follicles were dissected from 72 ovaries following ovariohysterectomies. MiRNAs regulating FSHR and ESR2 genes were selected from miRNA databases, and mature miRNA and mRNA expression profiling was performed using real-time polymerase chain reaction (PCR). The best miRNA for each target gene was selected considering the quantitative PCR (qPCR) performance and target prediction probability, selecting only miRNAs with a binding p-value of 1.0, and choosing cfa-miR-34a and cfa-let-7c for FSHR and ESR2, respectively. The expression levels comparing the different phases of the estrous cycle were evaluated using ANOVA. Pearson correlations between the expression pattern of each miRNA and their target genes were performed. Each miRNA and its target genes were expressed in the granulosa cells in all estrous phases. FSHR remained low in anestrus and proestrus, increased (p < 0.05) to the highest level in estrus, and decreased (p < 0.05) in diestrus. ESR2 showed the same trend as FSHR, with the highest (p < 0.05) expression in estrus and the lowest (p < 0.05) in anestrus and proestrus. A tendency for an inverse relationship was observed between the expression of miR-34a and FSHR only in the anestrus phase, while an inverse correlation (r = -0.8) was found between miRNA-7c and ESR2 (p < 0.01). The expression profile of miR-34a and miR-let-7c and their predicted target genes of dog ovarian follicles throughout the estrous cycle observed in this study suggest a role in the transcriptional regulation of FSHR and ESR2, which is the first evidence of the involvement of these miRNAs in the canine follicular function.

Chronic Undernutrition in Ovine Twin Pregnancies Abolishes Differences in Birth Weight Due to Sex: An Evaluation of the Role of Nutritional and Antioxidant Supplementation.

In twin pregnancies of discordant sex, the male fetus grows larger than the female co-twin. Our study aimed to determine the effect of the sex of co-twins on lambs' birth weight in ovine pregnancies developed under natural undernourishment. Additionally, we investigated whether the nutritional and/or antioxidant supplementation provided to ewes during pregnancy could modulate the potential effects associated with the sex of co-twins. Ninety-six birth records of twin pregnancies of sheep grazing the natural Patagonian prairies were analyzed. The animals were divided into four groups: control (no supplementation), N (concentrate supplementation, 100% NRC), A (antioxidant supplementation), and NA (concentrate + antioxidant supplementation). Supplementation occurred from day 35 of gestation onwards until lambing. There were no differences in female or male birth weight in the control undernourished group. However, in group N, females or males with sex-discordant co-twins had a higher birth weight than did those with co-twins of the same sex. Group A males with female co-twins had a higher birth weight compared to males whose co-twins were also males. In NA lambs, males had a higher birth weight compared to females, regardless of their co-twin's sex. Therefore, chronic undernutrition abolished the differences in birth weight due to fetal sex. Restoring maternal nutrition or antioxidant supplementation tends to normalize birth weight and restore the differences between females and males. This effect is enhanced with the combined supplementation of concentrated food and antioxidants.

Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium.

In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs.

Fertility in a high-altitude environment is compromised by luteal dysfunction: the relative roles of hypoxia and oxidative stress.

BACKGROUND: At high altitudes, hypoxia, oxidative stress or both compromise sheep fertility. In the present work, we tested the relative effect of short- or long-term exposure to high altitude hypobaric hypoxia and oxidative stress on corpora luteal structure and function. METHODS: The growth dynamics of the corpora lutea during the estrous cycle were studied daily by ultrasonography in cycling sheep that were either native or naïve to high-altitude conditions and that were supplemented or not supplemented with antioxidant vitamins. Arterial and venous blood samples were simultaneously drawn for determination of gases and oxidative stress biomarkers and progesterone measurement. On day five after ovulation in the next cycle, the ovaries were removed for immunodetection of luteal HIF-1alpha and VEGF and IGF-I and to detect IGF-II gene expression. RESULTS: The results showed that both short- and long-term exposure to high-altitude conditions decreased luteal growth and IGF-I and IGF-II gene expression but increased HIF-1 alpha and VEGF immunoexpression. The level of plasma progesterone was also increased at a high altitude, although an association with increased corpus luteum vascularization was only found in sheep native to a high-altitude location. Administration of antioxidant vitamins resulted in a limited effect, which was restricted to decreased expression of oxidative stress biomarkers and luteal HIF-1alpha and VEGF immunoexpression. CONCLUSIONS: Exposure of the sheep to high-altitude hypobaric hypoxia for short or long time periods affects the development and function of the corpus luteum. Moreover, the observed association of oxidative stress with hypoxia and the absence of any significant effect of antioxidant vitamins on most anatomical and functional corpus luteum traits suggests that the effects of high altitude on this ovarian structure are mainly mediated by hypoxia. Thus, these findings may help explain the decrease in sheep fertility at a high altitude.

Steroidogenesis in sheep pregnancy with intrauterine growth retardation by high-altitude hypoxia: effects of maternal altitudinal status and antioxidant treatment.

Sheep pregnancy in high-altitude environments frequently involves hypoxia and oxidative stress and causes intrauterine growth retardation. The adverse effects of altitude on fetal growth can be prevented by the administration of antioxidant vitamins, but the mechanisms responsible are not well known. The maintenance of a viable pregnancy depends largely on adequate placental steroidogenesis, especially in the last two-thirds of pregnancy. Thus, in the present study we evaluated the effect of antioxidant vitamins (C and E) on plasma concentrations of progesterone and 17ß-oestradiol during the last two-thirds of high-altitude pregnancies in ewes both native and naïve to the high-altitude environment. In addition, pregnancy outcomes were evaluated by determining the bodyweight of newborn lambs. Sex steroid patterns differed between ewes with and without vitamin supplementation. Concentrations of plasma progesterone and 17ß-oestradiol were significantly higher in the supplemented groups from approximately 40 days before parturition until near term. Newborn weights were significantly lower in animals not adapted to the higher altitude, and vitamin supplementation prevented this decrease. In conclusion, the administration of antioxidant vitamins in the present study enhanced placental steroidogenesis, thus favouring fetal development in pregnancies developing at high altitudes.

Meiotic Development of Canine Oocytes from Poly-Ovular and Mono-Ovular Follicles after In Vitro Maturation.

Poly-ovular follicles are defined as those with more than one oocyte present in single follicles. The occurrence frequency of this follicle type is higher in canines than that in other species. This study aimed to evaluate the in vitro meiotic maturation of dog oocytes from this follicle type in comparison to those from mono-ovular follicles of various sizes (small antral, medium antral, and large antral) considering different phases of the estrus cycle (anestrus, proestrus, estrus, and diestrus). Canine oocytes were obtained separately from the poly-ovular and mono-ovular antral follicles from the ovaries of adult females. In each experimental replicate, cumulus-oocyte complexes (COCs) from poly-ovular and mono-ovular follicles were incubated in supplemented TCM-199 at 38.5 °C and 5% CO2 for 72 h. After culturing, the meiotic development of each oocyte was evaluated using epifluorescence microscopy. Meiotic stages were classified into germinal vesicle (GV), germinal vesicle breakdown (GVBD), first metaphase (MI), and second metaphase (MII). Data were evaluated using an analysis of variance. Oocytes from poly-ovular follicles at all phases exhibited a higher (p < 0.05) percentage of oocytes arrested at the GV stage than those from mono-ovular follicles, showing the highest rate of GV in small antral follicles during anestrus. In contrast, there were no differences in MII rates (p < 0.05) in oocytes from mono-ovular and poly-ovular follicles during the estrus and diestrus phases in all sizes evaluated, with the highest MII rate in estrus. These results suggest that oocytes from poly-ovular follicles can resume meiosis at a slower rate than those from mono-ovular follicles; however, the maturation in vitro of such oocytes is possible. Furthermore, the relationship between the maturation capacity of oocytes from both poly-ovular and mono-ovular follicles depends on the ovarian cycle and follicular development.

Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC).

Although spermatogonial stem cells (SSC) constitute primary candidates for in vitro germ cell (GC) derivation, they are scarce and difficult to maintain in an undifferentiated state. Alternatively, mesenchymal stem cells (MSC) are also candidates for GC derivation due to their simplicity for culture and multipotential for transdifferentiation. The aim of the present study was to compare the GC differentiation potentials of bull peripheral blood-derived MSC (PB-MSC) and SSC using an in vitro 3D co-culture system with Sertoli cells (SC). Samples of PB-MSC or SSC co-cultures with SC were collected on days 0, 7, 14 and 21 and analyzed for pluripotency, GC and mesenchymal marker expression. Co-culture of PB-MSC+SC resulted in down-regulation of NANOG and up-regulation of OCT4 at day 7. In comparison, co-culture of SSC+SC resulted in consistent expression of NANOG, OCT4 and SOX2 at day 14. During co-culture, SSC+SC increased the expression of DAZL, PIWIL2, FRAGILIS and STELLA and activated the expression of STRA8, whereas co-culture of PB-MSC+SC only increased the expression of DAZL and PIWIL2. Thus, co-culture of bull PB-MSC+SC and SSC+SC in 3D SACS results in differential expression of pluripotency and GC markers, where bull SSC display a more robust GC differentiation profile compared to PB-MSC.

Effect of progesterone on in vitro meiotic maturation of canine oocytes associated with Cx37 and Cx43 gene expression.

Progesterone (P4) concentrations in canines are exceptionally high in the periovulatory period. However, the mechanisms by which P4 modulates final oocyte development in dogs remain to be characterized. The aim of this study was to evaluate the effect of P4 on meiotic development related to the gene expression of connexin 37 (Cx37) and connexin 43 (Cx43) in the canine cumlus oocyte complexes (COCs). COCs were isolated from 120 canine ovaries after a routine ovariohysterectomy. In each experiment, groups of COCs retrieved from the antral follicles were subjected to in vitro maturation (IVM) for 72 h without (control) or with P4 (50 µg/mL and 100 µg/mL) or the P4 receptor antagonist, aglepristone (RU534 at 1 µM and 10 µM). Some of the COCs recovered (from each group) after 72 h of IVM were subjected to meiotic evaluation; the remaining COCs, and those not subjected to IVM, were used to analyze the gene expression of Cx37 and Cx43 by qPCR. The results were evaluated using ANOVA. The addition of P4 increased (P < 0.05) the meiotic development compared to that in the control or aglepristone groups. The highest (P < 0.05) percentage of oocytes in the MII stage was observed upon P4 supplementation. In contrast, the highest percentage (P < 0.05) of oocytes arrested in the GV stage and the lowest (P < 0.05) percentages in the MII stage were observed for COCs cultured with aglepristone. Although a significant decrease in the mRNA levels of both connexins was observed after culturing, no effect on Cx37 and Cx43 gene expression was observed when exogenous P4 was added compared to those of the control group. However, COCs cultured with aglepristone exhibited higher (P < 0.05) expression of Cx37 and Cx43 than COCs in the control IVM-group, regardless of the concentration. In conclusion, our results suggest that a high dosage of P4 during IVM enhances the nuclear maturation of canine oocytes without altering the gene expression levels of Cx37 and Cx43. However, the increase in their expression upon treatment with a P4 antagonist indicates an in vivo role for this hormone in the endogenous modulation of both Cx37 and Cx43.

Colostrum traits and newborn body weight and growth: comparison between single and twin underfed sheep pregnancies.

Maternal nutrition during gestation plays an important role in colostrum production, postnatal growth, and survival of newborn lambs, especially in twin gestations. This research aimed to investigate the effects of chronic natural undernutrition on colostrum traits and early lamb's postnatal growth born from single and twin sheep pregnancies developed in a restrictive prairie, representative of southern Patagonia. Single- and twin-bearing ewes (n = 20 per group) were maintained grazing in a natural pasture. At 140 days of gestation, ewes were placed in individual pens for lambing control. Colostrum was collected immediately after delivery and at 12, 24, and 36 h postpartum, for determination of yield and composition. Maternal blood was obtained at 140 days of gestation and at lambing for plasma glucose, progesterone, 17ß-estradiol, and IgG determination. Newborn lamb blood for determining glycaemia and IgG was collected at birth and at 12, 24, 36, and 120 h after birth. Lamb mortality and growth was assessed from birth until 30 days of life. No differences were observed in progesterone and 17ß-estradiol. There were no differences in colostrum yields and fat components, however single- had higher values of protein and lactose than twin-bearing ewes (p < 0.05 for both). Singletons had higher glycaemia than twins at 12 h postpartum (102.2 ± 32.8 vs. 73.4 ± 29.9 mg/dL, p < 0.05). Colostrum IgG content was similar at delivery but higher in single ewes at 12 and 24 h, reaching a similar values at 36 h (4.7 ± 9.7 and 5.8 ± 7.7 mg/mL in single and twin pregnancies, respectively). Newborn IgG was higher in singletons compared to twins at least until 48 h of life. Lams body weight was always superior in singleton than twins from birth until 30 days of life. Mortality did not differ during the first week of life, but it increased significantly only in twins until day 30 of life. Undernourishment in pregnant ewes affected colostrum quantity and quality, resulting in a lower postnatal growth and a higher mortality in twins. Alternative managements favoring fetal growth, birth weight and neonatal viability in twin sheep pregnancies are needed, when flocks are breed under harsh environments.

Oxidative Stress and Fetal Growth Restriction Set Up Earlier in Undernourished Sheep Twin Pregnancies: Prevention with Antioxidant and Nutritional Supplementation.

Hypoxemia and oxidative stress, resulting in intrauterine growth restriction (IUGR) in undernourished twin sheep pregnancies, has been described in near-term studies. Our aim was to evaluate if the counteractive effects of maternal nutritional or antioxidant supplementation on the fetal redox status were evident before the accelerated fetal growth phase. Forty twin-bearing ewes grazing on natural Patagonian prairie were randomly assigned to four groups (n = 10 each; P: control ewes consuming mainly natural pasture; P+A: pasture plus antioxidants; P+C: pasture plus concentrate; P+A+C: pasture plus antioxidants and concentrate). Daily herbal antioxidants were supplemented in a feedstuff concentrate as a premix from day 35 until day 100 of gestation, when fetal venous cord blood samples and biometric measurements were obtained via cesarean section. The fetuses from group P were clearly hypoxemic. An analysis of variance showed that maternal antioxidant supplementation showed a trend of increased PO2, SatHb, and Ht, effects not observed in P+C fetuses. Antioxidants decreased the fetal MDA concentration (p < 0.05). Fetal TAC was increased by the antioxidants and concentrate (p < 0.05). Antioxidant supplementation showed a trend to increase fetal body weight but not biometry. The results suggest that negative effects of oxidative stress occur earlier than the overt growth arrest, and the maternal administration of antioxidants may constitute a good nutritional strategy for the early prevention of IUGR.

Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations.

Organoids are 3D-culture systems composed of tissue-specific primary cells that self-organize and self-renew, creating structures similar to those of their tissue of origin. Testicular organoids (TOs) may recreate conditions of the testicular niche in domestic and wild cattle; however, no previous TO studies have been reported in the bovine species. Thus, in the present study, we sought to generate and characterize bovine TOs derived from primary testicular cell populations including Leydig, Sertoli and peritubular myoid cells. Testicular cells were isolated from bovine testes and cultured in ultra-low attachment (ULA) plates and Matrigel. TOs were cultured in media supplemented from day 3 with 100 ng/mL of BMP4 and 10 ng/mL of FGF2 and from day 7 with 15 ng/mL of GDNF. Testicular cells were able to generate TOs after 3 days of culture. The cells positive for STAR (Leydig) and COL1A (peritubular myoid) decreased (p < 0.05), whereas cells positive for WT1 (Sertoli) increased (p < 0.05) in TOs during a 28-day culture period. The levels of testosterone in media increased (p < 0.05) at day 28 of culture. Thus, testicular cells isolated from bovine testes were able to generate TOs under in vitro conditions. These bovine TOs have steroidogenic activity characterized by the production of testosterone.

Effects of antioxidant vitamins on newborn and placental traits in gestations at high altitude: comparative study in high and low altitude native sheep.

The present study evaluated the hypothesis that the effects of hypoxia on sheep pregnancies at high altitude (HA) are mediated by oxidative stress and that antioxidant vitamins may prevent these effects. Both HA native and newcomer ewes were maintained at an altitude of 3,589 m during mating and pregnancy. Control low altitude (LA) native ewes were maintained at sea level. Half of each group received daily oral supplements of vitamins C (500 mg) and E (350 IU) during mating and gestation. Near term, maternal plasma vitamin levels and oxidative stress biomarkers were measured. At delivery, lambs were weighed and measured, and placentas were recovered for macroscopic and microscopic evaluation. Vitamin concentrations in supplemented ewes were two- or threefold greater than in non-supplemented ewes. Plasma carbonyls and malondialdehyde in non-supplemented ewes were consistent with a state of oxidative stress, which was prevented by vitamin supplementation. Vitamin supplementation increased lamb birthweight and cotyledon number in both HA native and newcomer ewes, although placental weight and cotyledon surface were diminished. Placentas from vitamin-supplemented HA ewes were similar to those from ewes at sea level, making these placental traits (weight, number and diameter of cotyledons) similar to those from ewes at sea level. Vitamin supplementation had no effect on LA pregnancies. In conclusion, supplementation with vitamins C and E during pregnancy at HA prevents oxidative stress, improving pregnancy outcomes.

Acrosin release and acrosin activity during incubation in capacitating media using fresh and frozen-thawed dog sperm.

We evaluated the effect of time and temperature on acrosin release from the acrosomal cap and the activity of this enzyme during in vitro capacitation in fresh and frozen/thawed dog sperm. Sperm-rich fractions of six ejaculates from three dogs were processed as fresh and frozen samples. Each sperm sample was incubated in canine capacitation medium (CCM) for 0, 1, 2 and 3 h at 20°C and at 37°C. After incubation, the samples were assessed by the indirect immunofluorescent staining technique. The probability of having unlabeled sperm (PUS), indicating acrosin loss, was modelled by a binomial distribution using logistic regression. There was a linear relationship between PUS and time at both temperatures (p<0.001); however, a major percentage of unlabeled sperm was observed in frozen/thawed samples soon after incubation, indicating that the release of acrosin was affected by capacitation time, mainly in frozen samples. Temperature influenced acrosin release only in cryopreserved sperm (p<0.05). Acrosin activity was measured by digestion halos on slides coated with gelatin-substrate film during each time period; a significant increase in the number of large halos was observed in fresh samples throughout the experiment, whereas frozen/thawed sperm showed a decreased rate of halo diameters during culture. Thus, there appears to differences between fresh and frozen dog sperm in terms of acrosin release and the level of acrosin activity in the course of in vitro capacitation.

New Insights in Canine Reproduction.

Reproduction in canids has been characterized by having some peculiar aspects, such as the extended reproductive cycle and ovulation of immature oocytes [...].

Expression Profiles of the Progesterone Receptor, Cyclooxygenase-2, Growth Differentiation Factor 9, and Bone Morphogenetic Protein 15 Transcripts in the Canine Oviducts during the Oestrous Cycle.

The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs.