In vitro differentiation of bovine bone marrow-derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors.

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFß1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFß1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21-day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFß1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.

Safety and efficacy of a mesenchymal stem cell intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis.

Although, antibiotics are effective in the treatment of bovine mastitis, they do not address the regeneration of mammary glandular tissue and have been associated to the increment in antimicrobial resistance worldwide. Considering the necessity of alternative therapies for this disease of high economic impact and the reported regenerative and antibacterial effects of mesenchymal stem cell (MSCs), we evaluated the safety and efficacy of an allogenic MSC-based intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis. In a safety trial, heifers were inoculated intramammarily with a 2.5 × 107-suspension of bovine fetal AT-MSCs on experimental days 1 and 10. Animals were evaluated clinically on a daily basis during a 20-day experimental period and blood samples were collected for hemogram determination and peripheral blood leukocytes (PBLs) isolation. In an efficacy trial, Holstein Friesian cows were inoculated with S. aureus and treated intramammarily with vehicle (NEG; days 4 and 10), antibiotics (ATB; days 4 and 5) or a suspension of 2.5 × 107 AT-MSCs (MSC; days 4 and 5). Cows were clinically evaluated daily and milk samples were collected for somatic cell count (SCC) and colony forming units (CFU). Blood samples were collected for serum haptoglobin and amyloid A determination. Intramammary administration of two doses of bovine fetal AT-MSCs in healthy cows did not induce changes in clinical or hematological variables, and gene expression profiles in PBLs associated to activation (CD4, CD8, CD25, CD62L and CD69) and proinflammatory cytokines (CCL2, CCL5, IL2, CXCL3, IFNγ, and TNFα). Quarters of MSC group of cows had similar SCC log/mL in milk compared to infected quarters of ATB or NEG cows. However, quarters of MSC cows had lower CFU log/mL in milk compared to quarters of NEG cows. Intramammarily inoculation of repeated doses of 2.5 × 107 allogenic AT-MSCs did not induce clinical or immunological response in healthy cows. Moreover, MSC-intramammary treatment reduced bacterial count in milk of cows with S. aureus clinical mastitis compared to untreated cows. This work provides initial evidence for the safety and efficacy of an allogenic MSC-based intramammary therapy for the treatment of bovine mastitis.

Comparative analysis of in vitro proliferative, migratory and pro-angiogenic potentials of bovine fetal mesenchymal stem cells derived from bone marrow and adipose tissue.

Mesenchymal stem cells (MSCs) are found in virtually all tissues, where they self-renew and differentiate into multiple cell types. Cumulative data indicate that MSCs secrete paracrine factors that may play key roles in the treatment of various acute and chronic pathological conditions in diverse animal species including cattle. The aim of the present study was to compare the potentials for proliferation, migration and pro-angiogenesis of bovine fetal BM-MSCs and AT-MSCs under in vitro conditions. Growth curves and population doubling time (PDT) were determined for BM-MSCs and AT-MSCs in order to compare in vitro cell proliferation potentials. The ability of BM-MSCs and AT-MSCs to migrate was evaluated by scratch plate and transwell migration assays. The pro-angiogenic potential of conditioned medium from BM-MSCs and AT-MSCs was compared using an endothelial cell (EC) tubule formation assay. BM-MSCs displayed higher proliferation curves and doubled their populations in fewer days compared to AT-MSCs. No significant differences were detected in the number of migrant cells between BM-MSCs and AT-MSCs; however, a higher migration value was detected for BM-MSCs compared to fibroblasts (FBs), and a higher number of migrant cells were attracted by DMEM supplemented with 5% fetal bovine serum (FBS) compared to stromal cell-derived factor-1 (SDF-1). More tubules of ECs were formed after exposure to concentrated conditioned medium from AT-MSCs compared to BM-MSCs, FBs or DMEM controls. Despite common mesodermal origin, BM-MSCs display higher proliferative capacity and lower pro-angiogenic potential compared to AT-MSCs; however, both cell types possess similar migratory ability.

Sertoli cell-mediated differentiation of bovine fetal mesenchymal stem cells into germ cell lineage using an in vitro co-culture system.

In vitro gamete derivation based on differentiation of germ cells (GC) from stem cells has emerged as a potential new strategy for the treatment of male infertility. This technology also has potential applications in animal reproduction as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to differentiate into mesodermal lineages. Under the effect of selected bioactive factors, MSC upregulate expression of pluripotent and GC specific-markers revealing their potential for GC differentiation. In addition to the effect of trophic factors, cell-to-cell interaction with Sertoli cells (SC) may be required to guide the sequential differentiation of MSC into GC. Thus, the aim of the present study was to investigate the effect of coculture with SC on the potential for in vitro GC differentiation of bovine fetal MSC (bfMSC) derived from bone marrow (BM-MSC) and adipose tissue (AT-MSC). bfMSC were isolated from male bovine fetuses and SC were collected from adult bull testes. The effect of SC interaction with BM-MSC or AT-MSC was analyzed on the expression of pluripotent factors OCT4 and NANOG, GC genes FRAGILLIS, STELLA and VASA and male GC markers DAZL, PIWIL2, STRA8 and SCP3 at Day 14 of coculture. Flow cytometry analyses detected that the majority (95,5% ±â€¯2.5; P < 0.05) of the isolated population of SC cultures were positive for SC-specific marker WT1. Levels of mRNA of WT1 in BM-MSC and AT-MSC were lower (P < 0.05) compared to SC; whereas, WT1 expression was not detected in bovine fetal fibroblasts (FB). Cocultures of BM-MSC and AT-MSC with SC had higher (P < 0.05) OCT4 mRNA levels compared to monocultures of BM-MSC, AT-MSC and SC. Moreover, cocultures of BM-MSC with SC had higher (P < 0.05) proportion of cells positive for Oct4 and Nanog compared to monocultures of BM-MSC and SC. Levels of mRNA of DAZL, PIWIL2 and SCP3 were upregulated in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Accordingly, the proportion of cells positive for Dazl were higher (P < 0.05) in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Changes in gene expression profiles during coculture of SC with AT-MSC suggest that cell-to-cell interaction or bioactive factors provided by SC may induce progression of AT-MSC into early stages of GC differentiation.

Ram semen deterioration by short-term exposure to high altitude is prevented by improvement of antioxidant status.

Ovine reproduction efficiency in herds at high altitude (ha) is lower than that at low altitude (la). In ewes, ha effects are due to hypoxia and oxidative stress. Our aim was to establish the effect of antioxidant vitamin supplementation on semen traits and antioxidant status of rams exposed to short or long time ha. A total of 32 rams native to la (~500 m) were used, 16 were kept at la and the other 16 were brought to ha (~3600 m), where they were placed in the same flock as the ha native rams (n=16). Half of the animals in each group were supplemented daily with vitamins C 600 mg and E 450 IU per os, during the entire experimental period, starting the 4th day after animal's arrival at ha (day 0). At days 0, 30 and 60 of treatment, blood and semen samples were collected for evaluation of antioxidant status and semen standard characteristics. Data were compared within each experimental time by analysis of variance using a general linear model. Elevated concentrations of oxidative stress biomarkers were present in blood from animals maintained at ha. Ejaculates from ha exposed rams showed decreased sperm concentration, progressive motility and viability, in addition to decreased antioxidant status in seminal fluid. A total of 30 days of oral supplementation with vitamins C and E prevented some ha negative effects on semen characteristics, mainly in recently ha exposed rams. It is concluded that exposure of rams to ha negatively affects semen quality, where oxidative stress plays a predominant role. These effects are mainly prevented by oral supplementation of vitamins C and E, which constitutes a simple and cheap alternative to improve semen quality of rams when they are moved to ha.

Tissue localization of GM-CSF receptor in bovine ovarian follicles and its role on glucose uptake by mural granulosa cells.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and ß-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and ß-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3ß-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake.

Comparison of three estrus detection systems during summer in a large commercial dairy herd.

The objective of the study was to compare three systems for estrus detection and combinations of these systems on a large commercial dairy (1075 lactating cows) during stress of summer heat. At 37-45 days in milk (DIM), 255 cows were fitted with a HeatWatch device (HW; DDx Inc., Denver, CO), an activity sensor ALPRO (ALPRO; DeLaval Inc., Kansas City, MO), and visually observed (VO) three times daily. Pregnancy status was determined by uterine palpation per rectum 35-49 days following artificial insemination (AI). Effects of DIM, parity, standing events, inseminator, and interval between onset of estrus and AI on conception rates were determined using logistic regression. Efficiencies for detection of estrus, determined by comparing detected periods of estrus with a theoretical total of 570 periods, were 49.3% (VO), 37.2% (ALPRO), 48.0% (HW), and 80.2% for all three systems simultaneously. Conception rates (LSM+/-S.E.) for cows detected by one or more of the three systems were 6.2+/-3.9 for VO, 19.8+/-5.6 for ALPRO, 17.3+/-5.0 for HW, 22.8+/-7.0 for VO+ALPRO, 26.9+/-4.6 for VO+HW, 23.2+/-5.2 for ALPRO+HW, and 18.4+/-4.7 for VO+ALPRO+HW. Inseminations performed during no and mild heat stress (temperature-humidity index; THI< or =76) had greater conception rate (P<0.05; 38.8%) compared to AI performed during moderated heat stress conditions (THI>76; 17.6%). Number of mounts were higher for primiparous versus multiparous cows (P<0.05). Cows over 80 DIM during estrus exhibited fewer (P<0.05) standing events. The highest conception rate occurred with the combination of VO+HW, which confirms the premise that combination of multiple systems enhances both the efficiency and accuracy of estrus detection.

GFP labelling and epigenetic enzyme expression of bone marrow-derived mesenchymal stem cells from bovine foetuses.

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Since MSC from adult tissues represent a promising source of cells for a wide range of cellular therapies, there is high scientific interest in better understanding the potential for genetic modification and the mechanism underlying differentiation. The main objective of this study was to evaluate the potential for gene delivery using a GFP vector and lipofectamine, and to quantify the expression of epigenetic enzymes during foetal bMSC multilineage differentiation. Proportion of GFP-positive cells achieved (15.7% ± 3.5) indicated moderately low transfection efficiency. Analysis of DNA methyltransferase expression during MSC multilineage differentiation suggested no association with osteogenic and chondrogenic differentiation. However, up-regulation of KDM6B expression during osteogenic differentiation was associated with adoption of osteogenic lineage. Furthermore, increase in epigenetic enzyme expression suggested an intense epigenetic regulation during adipogenic differentiation.

Transvaginal ultrasound-guided cumulus oocyte complexes aspiration and in vitro embryo production in suckled beef and lactating dairy cattle on pasture-based management conditions.

This study was conducted to determine the use of repeated transvaginal ultrasound-guided cumulus oocyte complex (COC) aspiration on COC recovery rate, in vitro embryo production (IVP) and subsequent pregnancy rates in Holstein Friesian (HF) and Aberdeen Angus (AA) cows (Experiment 1), and in pregnant and non-pregnant Holstein Friesian cows (Experiment 2). Cycling, non-pregnant HF (n=17) and AA (n=32) cows with 40-70 days postpartum, between 3 and 5 years of age were used in the Experiment 1. All cows were submitted to repeated transvaginal ultrasound-guided COC aspiration twice a week for 5-7 weeks. Cumulus ooctye complexes (COC) were in vitro matured, fertilized and cultured for 8 days. An overall of 100 and 350 embryos from HF and AA cows respectively were cryopreserved using a conventional slow freezing (Experiment 1). A total of 81 and 285 frozen-thawed embryos from HF and AA cows respectively were transferred to recipient cows. Pregnancy diagnosis was performed at 60 and 150 days of gestation using transrectal ultrasonography. In Experiment 2, cycling non-pregnant (n=9) and pregnant (n=8) HF cows were submitted to repeated ultrasound-guided COC aspiration and COC were in vitro matured, fertilized and cultured as in Experiment 1, except that embryos were cryopreserved but not thawed and transferred as described for Experiment 1. The results of this study indicate that COC recovery rate and blastocyts production are affected by the breed of the donor cow. The quality of blastocyts produced from both breed did not differ in terms of pregnancy and calving rates (Experiment 1). The physiologic state of pregnancy did not affect COC recovery rate and blastocysts production per donor/session (Experiment 2). The use of ultrasound-guided COC aspiration and IVP could be a powerful technique to improve the genetic of beef and dairy cattle managed under pasture-based conditions management in the southern Chile.