A bicentric cohort study comparing umbilical cord and neonatal blood samples for chemistry tests at birth.

OBJECTIVES: To compare umbilical cord and neonatal blood for chemistry tests upon admission to the neonatal intensive care unit (NICU). METHODS: We designed a prospective, bicentric cohort study enrolling newborns (n = 71) with a planned admission to the NICU. Paired samples of umbilical cord and infant's blood were collected, analyzed, and compared. An intraclass correlation coefficient (ICC) was calculated for a repeatability analysis, and a Bland-Altman analysis was performed to assess the agreement between the 2 methods of sampling. The multivariable coefficient of determination (R2) was reported to quantify the degree of correlation between the methods of measurement. RESULTS: The degree of agreement between the 2 sampling methods for chemistry tests was fair to good for high-sensitivity C-reactive protein (ICC = 0.79 [95% CI, 0.67-0.87]), phosphorus (ICC = 0.83 [95% CI, 0.73-0.90]), and albumin (ICC = 0.76 [95% CI, 0.60-0.86]), while it was good to excellent for γ-glutamyl transpeptidase (ICC = 0.95 [95% CI, 0.88-0.98]) and procalcitonin (ICC = 0.90 [95% CI, 0.76-0.96]). CONCLUSIONS: Umbilical cord blood is a reliable replacement source for multiple chemistry tests at birth. This sampling method has the potential to minimize the risk of transfusion-requiring anemia in newborns and its associated complications. Further studies are warranted to assess the efficacy of this strategy in improving neonatal outcomes.

Bone morphogenetic protein 15 intrafollicular injection inhibits ovulation in cattle.

Oocyte-derived bone morphogenetic protein 15 (BMP15) is one of the main local regulators of ovarian physiology, but its role in the regulation of preovulatory follicles and ovulation is not well established. Therefore, this study was conceived to determine the effect of intrafollicular injection (IFI) of BMP15 on final follicular growth, ovulation and luteinization in cattle. Initially, it was observed that relative mRNA abundance of the BMP15 receptor BMPR1B in granulosa cells was regulated by GnRH treatment, and it was negatively correlated (R2 = 0.5; P < 0.001) to progesterone concentration in follicular fluid (FF) from preovulatory follicles. The IFI of recombinant human BMP15 tended to inhibit the growth of dominant follicles, as evidenced by an average increase of only 7.7% in the follicular diameter (from 8.8 mm to 9.1 mm) at 36 h post injection compared to 36.4% increase (from 8.9 mm to 14 mm) in the control group. Injection of BMP15 into preovulatory follicles (12-14 mm), simultaneously to im GnRH treatment, inhibited ovulation compared to control group, but did not prevent luteinization and progesterone production. Most of preovulatory follicles injected with BMP15 became luteinized cysts. Collectively, these findings indicate a suppressive role of BMP15 on later follicular development and ovulation in cattle, but not on luteogenesis and progesterone secretion.

Expression of steroidogenic enzymes and TGFß superfamily members in follicular cells of prepubertal gilts with distinct endocrine profiles.

Regulation of the transforming growth factor beta (TGFß) superfamily by gonadotrophins in swine follicular cells is not fully understood. This study evaluated the expression of steroidogenic enzymes and members of the TGFß superfamily in prepubertal gilts allocated to three treatments: 1200 IU eCG at D -3 (eCG); 1200 IU eCG at D -6 plus 500 IU hCG at D -3 (eCG + hCG); and the control, composed of untreated gilts. Blood samples and ovaries were collected at slaughter (D0) and follicular cells were recovered thereafter. Relative gene expression was determined by real-time PCR. Serum progesterone levels were greater in the eCG + hCG group compared with the other groups (P < 0.01). No differences were observed in the expression of BMP15, BMPR1A, BMPR2, FSHR, GDF9, LHCGR and TGFBR1 (P > 0.05). Gilts from the eCG group presented numerically greater mean expression of CYP11A1 mRNA than in the control group that approached statistical significance (P = 0.08) and greater expression of CYP19A1 than in both the eCG and the control groups (P < 0.05). Expression of BMPR1B was lower in the eCG + hCG treatment group compared with the control (P < 0.05). In conclusion, eCG treatment increased the relative expression of steroidogenic enzymes, whereas treatment with eCG + hCG increased serum progesterone levels. Although most of the evaluated TGFß members were not regulated after gonadotrophin treatment, the downregulation of BMPR1B observed after treatment with eCG + hCG and suggests a role in luteinization regulation.

Regulation and function of leptin during ovarian follicular development in cows.

Although it is well documented that leptin signals the body nutritional status to the brain, mechanisms of leptin regulation at the ovary are not well understood. This study was conducted to determine whether there was leptin and the receptor for leptin (LEPR) in cattle ovarian follicles and to investigate potential actions of leptin on follicular growth in vivo and on regulation of granulosa cell functions in vitro. There was leptin and LEPR in granulosa and theca cells of dominant and subordinate follicles, with greater immunostaining for leptin in granulosa cells of subordinate follicles. There was a lesser relative abundance of leptin receptor gene-related protein (LEPROT) and of the adiponectin receptors 1 (ADIPOR1) and 2 (ADIPOR2) mRNA transcripts in granulosa cells of subordinate than dominant follicles (P < 0.05). Intrafollicular injection of either 100 or 1000 ng/mL leptin did not affect the diameter and the growth of dominant follicles (P> 0.05). Supplementation of in vitro culture medium with different leptin concentations did not affect (P > 0.05) the relative abundance of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), signal transducer and activator of transcription 3 (STAT3) and X-linked inhibitor of apoptosis protein (XIAP) mRNA transcripts in granulosa cells. These findings indicate that leptin and LEPR are present in the follicular cells of cattle ovaries, but leptin apparently does not have essential functions in steroidogenesis and growth of dominant follicles.

Transforming growth factor-beta family members are regulated during induced luteolysis in cattle.

The transforming growth factors beta (TGFß) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFß family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P<0.05) in comparison to 0 h. The relative mRNA abundance of BMP1, BMP2, BMP3, BMP4, BMP6, ACVR1B, INHBA and INHBB was upregulated up to 12 h post PGF (P<0.05). On the other hand, TGFBR3 mRNA that codes for a reservoir of ligands that bind to TGF-beta receptors, was lower at 48 h. In conclusion, findings from this study demonstrated that genes encoding several TGFß family members are expressed in a time-specific manner after PGF administration.

Oncostatin M and its receptors mRNA regulation in bovine granulosa and luteal cells.

Oncostatin M (OSM) and its receptor (OSMR) are members of the interleukin-6 family cytokines. Although OSM and OSMR expression was detected in human ovaries, their function and regulation during follicle development, ovulation and luteolysis have not been studied in any species. The aim of the present study was to investigate the levels of OSM and OSMR mRNA in bovine ovaries and the effect of OSM treatment on cultured granulosa cells. OSM mRNA was not detected in granulosa cells obtained from follicles around the time of follicular deviation and from pre-ovulatory follicles, whereas OSMR transcript levels were greater in granulosa cells of atretic subordinate follicles (P < 0.001). Abundance of OSMR mRNA increased in granulosa cells of preovulatory follicles, collected at 12 and 24 h after the ovulatory stimulus with gonadotropins (P < 0.001). In the luteal tissue, OSM mRNA abundance levels were higher at 24-48 h after PGF-induced luteolysis (P < 0.01) compared to 0 h, whereas OSMR mRNA was transiently increased at 2 h after PGF treatment (P < 0.05). In cultured granulosa cells, 10 ng/mL OSM in the presence of FSH increased BAX/BCL2 mRNA ratio (P < 0.05) compared to the control. Moreover, 100 ng/mL OSM in the presence of FSH increased OSMR (P < 0.05) and decreased XIAP mRNA (P < 0.05) levels, compared to the control group. These findings provide the first evidence that OSMR is regulated during follicle atresia, ovulation and luteolysis, and that OSM from other cells may mediate granulosa and luteal cell function, regulating the expression of genes involved in cell's viability.

Effects of supplying omega-3 polyunsaturated fatty acids to gilts after weaning on metabolism and ovarian gene expression.

Omega-3 PUFA may benefit sow reproductive performance, but effects on weaned gilts are unknown. This study evaluated the effects of supplementing omega-3 PUFA to gilts after weaning on growth, metabolic markers, and gene expression of steroidogenic enzymes and hormone receptors. For 52 d, gilts in the control group were fed 100 g/d of regular diets, whereas gilts in the omega-3 group were fed 75 g/d of such diets plus 25 g/d of the microalgae Schizochytium sp. (3.5 g/d of omega-3 PUFA; n = 8 gilts/group). Blood samples were collected at day 0, day 21, and day 52. Total serum cholesterol levels were lower for the omega-3 group than for the control group (P < 0.05), but high-density lipoprotein-cholesterol levels were reduced at day 52 for both groups (P < 0.05). Gilts in the omega-3 group presented lower feed intake, better feed conversion, and less-intense immunolabeling for leptin and its receptor in the cytoplasm of oocytes included in primordial/primary follicles than gilts in the control group (P < 0.05). The expression of genes coding for cholesterol side-chain cleavage and aromatase enzymes and the LH receptor in follicular cells was lower for supplemented gilts (P < 0.05). Compared with controls, supplemented gilts presented decreased serum cholesterol levels and better feed conversion, but leptin presence and gene expression for steroidogenic enzymes and for the LH receptor were lower at ovarian level.

Prepartum progestagen supplementation in swine: a strategy to facilitate piglet care and prevent early parturition / Suplementação de progestágenos no pré-parto em suínos: estratégia para facilitar assistência aos leitões e evitar partos precoces

ABSTRACT: Gestation length in swine has a considerable amplitude and both early and delayed parturition are common. This variation increases the occurrence of unassisted farrowing and could lead to a wide-ranging age at weaning for piglets born from one batch. Supervision of sow parturition is crucial to reduce mortality of neonate piglets. To facilitate assistance, induction of farrowing using prostaglandin F2α (PGF) has been widely used in batch farrowing systems, whereby synchronization would concentrate the time of farrowing, allowing for better organization of employees. However, a viable alternative method that can be implemented to manage farrowing is to sustain high progestagen levels in the final days of gestation and, consequently, prevent early parturition. Efficient techniques to delay farrowing such as using oral progestagen supplementation have been previously described, but are only recently being considered for commercial use. The present manuscript reviews publications regarding delaying parturition and discusses the use of intravaginal devices (IVDs) containing progestagen. There is limited data addressing the effect of progestagen treatment during gestation on productive and reproductive performance. Therefore, future studies should focus on improving synchronization protocols following progestagen supplementation and evaluating piglet viability and sow fertility, before widely using progestagen supplementation to manipulate parturition.

RESUMO: Como a duração da gestação de suínos pode ter ampla variação, é comum a ocorrência de partos antecipados ou gestações prolongadas. Isso aumenta as chances de partos sem assistência e leva a uma grande variação de idade dos leitões dentro do lote de produção. Portanto, a supervisão do parto é indispensável para reduzir as perdas neonatais. Para facilitar o auxílio aos leitões, a indução do parto com prostaglandina F2α (PGF) é eficaz e amplamente utilizada, sendo indicada para concentrar os partos em momentos mais adequados, preferencialmente durante o horário com maior disponibilidade de colaboradores. Uma alternativa viável é manipular o momento do parto, através da manutenção de níveis plasmáticos elevados de progestágeno durante o final da gestação, a fim de evitar partos antecipados. Formas eficientes de evitar o parto através de suplementação oral de progestágenos foram descritas há décadas, mas apenas recentemente tem sido cogitada a utilização comercial. A presente revisão aborda estudos disponíveis na literatura relacionados ao protelamento do parto, incluindo a utilização de dispositivos intravaginais (DIVs) impregnados com progestágeno. São poucos os dados disponíveis relacionados ao uso de progestágenos na gestação com índices produtivos e reprodutivos. Portanto, alguns pontos ainda devem ser melhor avaliados, especialmente com relação à determinação da sincronia dos partos após o fim da suplementação com progestágenos, à viabilidade dos neonatos e à fertilidade subsequente das fêmeas antes da ampla adoção desta técnica.

Chemical castration in cattle with intratesticular injection of sodium chloride: Effects on stress and inflammatory markers.

Intratesticular injection (ITI) of sodium chloride (NaCl) is efficient for chemical castration of young calves, but its effects on calves welfare are unknown. Two experiments were conducted to evaluate the effects of ITI of 20% NaCl on stress and inflammatory markers in calves less than 20 days old and to assess the efficiency of ITI of 30% NaCl in 5 months old calves. In Experiment 1, control calves were only restrained and compared to calves submitted to castration through surgery (SC) and ITI with 20% NaCl (n = 9/group). No differences were observed for the eye corner temperature measured by thermography from 60 s before to 60 s after the procedures (P > 0.05). In the SC group, acute serum cortisol levels increased at 30 and 60 min after the procedure, but increased levels in the ITI group occurred only at 30 min (P < 0.05). Chronic discomfort markers were measured at 0, 24, 48, 72 and 96 h after the procedures (D0, D1, D2, D3 and D4, respectively). The serum levels of the paraoxonase 1 (PON1) enzyme and cortisol did not differ among groups (P > 0.05). Scrotal temperature was higher at D1 in the SC group than for the other groups, but lowest at D4 compared to the control (both P < 0.05). In Experiment 2, histological sections of testes were compared after ITI with either 30% NaCl or 30% calcium chloride (CaCl2), to intact calves (control). After 60 days, intact seminiferous tubules and mediastinum were observed after ITI with 30% NaCl, whereas coagulative necrosis, inflammatory infiltration and calcification occurred after ITI with 30% CaCl2. Efficient chemical castration through ITI of 20% NaCl in young calves was followed by slight stress and inflammatory responses compared to surgical castration. However, ITI of 30% NaCl was ineffective for chemical castration of 5 months old calves.

Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation.

In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25µM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.

Cord blood CD133 cells define an OV6-positive population that can be differentiated in vitro into engraftable bipotent hepatic progenitors.

Cell therapy represents the most promising alternative strategy for end-stage liver diseases and hepatic progenitors are the best candidates. We have identified a reservoir of immature hepatic precursors within human cord blood, which can derive engraftable bipotent progenitors. We isolated a stem cell subset CD133+/CD34+/OV6(low) expressing a surface-marker profile consistent with that of fetal liver cells. Upon induction of hepatic commitment by a medium containing cytokines and factors involved in vivo oval-cell activation, a heterogeneous cell population displaying characteristics of functional oval-cell-like bipotent hepatic progenitors was obtained. The cells expressed markers of hepatocytes and cholangiocytes and were highly enriched in OV6, c-Met, c-Kit, and Thy-1. They also displayed liver functional activity as glycogen storage, urea production, albumin secretion, and inducible CyP2B6 activity. When injected into liver-damaged severe-combined immunodeficient mice, induced bipotent hepatic progenitors appropriately engrafted livers of recipient animals, where they formed clusters of human-derived cells expressing human leucocyte antigen-class I, Hep-Par1, and OV6 antigens. Human-specific albumin, alpha-fetoprotein, and cytokeratin 19 were also expressed. In transplanted animals, AST serum levels showed a significative reduction with regard to controls. This human model for in vitro progenitor-cell activation may provide a powerful tool for elucidating the pathways and synergies that regulate this complex process and can represent a valuable source, exploitable for liver cell-based therapies and regenerative medicine.