Leptin decreases apoptosis and promotes the activation of primordial follicles through the phosphatidylinositol-3-kinase/protein kinase B pathway in cultured ovine ovarian tissue.

This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.

Amburana cearensis leaf extract maintains survival and promotes in vitro development of ovine secondary follicles.

The antioxidant properties of Amburana cearensis extract may be a useful substitute for standard cell culture medium. Thus, the aim of this study was to evaluate the effect of this extract, with or without supplementation, on in vitro survival and development of sheep isolated secondary follicles. After collection of the ovaries, secondary follicles were isolated and cultured for 18 days in α-MEM+ supplemented with bovine serum albumin, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (control medium) or into medium composed of different concentrations of A. cearensis extract without supplements (Amb 0.1; 0.2 or 0.4 mg/ml) or A. cearensis extract supplemented with the same substances described above for α-MEM+ supplementation. The A. cearensis supplemented medium was named Amb 0.1+; 0.2+ or 0.4+ mg/ml. There were more morphologically normal follicles in Amb 0.1 or Amb 0.4 mg/ml than in the control medium (α-MEM+) after 18 days of culture. Moreover, the percentage of antrum formation was significantly higher in Amb 0.1 or Amb 0.2 mg/ml than in α-MEM+ and Amb 0.1+ mg/ml, and similar to the other treatments. All A. cearensis extract media induced a progressive and significant increase in follicular diameter throughout the culture period. In conclusion, this study showed that 0.1 mg/ml of this extract, without supplementation, maintains follicular survival and promotes the development of ovine isolated secondary follicles in vitro. This extract can be an alternative culture medium for preantral follicle development.

Protein localization of epidermal growth factor in sheep ovaries and improvement of follicle survival and antrum formation in vitro.

The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre-antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18 days in α-MEM(+) (control) alone or supplemented with EGF (1, 10 or 50 ng/ml). The immunostaining for EGF was observed in oocytes from pre-antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18 days, the results showed that treatment with 50 ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α-MEM(+) ) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1 ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50 ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18 days of in vitro culture.

Immunolocalization of melatonin and follicle-stimulating hormone receptors in caprine ovaries and their effects during in vitro development of isolated pre-antral follicles.

The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre-antral follicles were evaluated. Follicles (≤200 µm) were cultured for 12 days in α-MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre-antral follicles.

Effects of condensed tannin-amended cassava silage blend diets on feeding behavior, digestibility, nitrogen balance, milk yield and milk composition in dairy goats.

Condensed tannins (CTs) are phenolic compounds derived from secondary plant metabolism that act as part of the plant's chemical defense system against pathogen invasion and herbivorous attack. This study aimed to evaluate the intake, digestibility, nitrogen (N) balance, production and composition of milk from goats fed cassava silage with added levels of CTs. Eight Anglo-Nubian goats with a mean BW of 40 ±â€¯2.0 kg were distributed in a double Latin square design with four levels of CTs (0, 25, 50 and 75 g/kg DM) with four 20-day periods with 15 days of adaptation and five evaluation days for each period. No differences were observed in DM, NDF, CP intake and feed conversion (grams of DM intake (DMI) per gram of milk produced); however, when expressed as percent of BW, DMI showed a quadratic increase to 29.1 g/kg. As the level of supplemented CTs increased in the diet, the CP digestibility (P = 0.023), NDF (P = 0.044), non-fiber carbohydrates (NFC; P = 0.032) and total digestible nutrients (P = 0.033) exhibited a linear decrease. Furthermore, the addition of CTs to cassava silage induced a linear increase in N-fecal excretion (P = 0.014) and a positive quadratic effect on N-retained (P = 0.014) and N-balance (P = 0.024) as well as a positive quadratic trend in N-digested (P = 0.092). Milk urea N (P = 0.023) decreased linearly. The addition of CTs to cassava silage had a positive quadratic effect on ruminating time (P = 0.011). In addition, comparing the use or non-use from the orthogonal contrast test, the inclusion of CTs in goat diet increased water and N-intake, CP and NDF digestibility, spent time eating and ruminating and N-balance and decreased milk production corrected3.5%, fat milk content, milk urea N and dry defatted extract of milk. Thus, adding CTs to cassava silage at 25 g/kg total DM promoted goats' greater use of the diet without impairing feed conversion and the quality of goat milk produced. Dietary levels of 50 and 75 g/kg total DM are not recommended because under the conditions of this study, they reduced the productive efficiency of dairy goats.

Apparent digestibility, nitrogen balance, and ruminal constituents in ram lambs fed high-concentrate diets containing soybean hulls.

Corn often constitutes the main portion of high-concentrate diets fed to feedlot lambs. However, soybean hulls (SH) may be used to replace corn, and may reduce feed costs and the risk of acidosis. The objective of this experiment was to determine the effect of SH inclusion rate in high-concentrate diets on DMI, apparent digestibility of nutrients, N balance, and some rumen constituents. Sixteen ruminally fistulated Santa Inês ram lambs (44.3 ± 5 kg of BW and 6 mo old) were housed in suspended metabolism crates. A randomized complete block design experiment with 4 diets and 4 blocks was used. Blocks were defined by BW, and diets were allotted randomly within block. The control diet contained 70% corn and 0% SH. In the remaining diets, SH replaced corn at the rate of 15, 30, or 45% of the original corn concentration, which resulted in 0, 10.5, 21.0, and 31.4% of SH in the dietary DM. The DMI (kg/d) increased linearly (P < 0.01) with increasing dietary SH inclusion. A trend was observed for linear decreases (P = 0.10) in apparent DM, OM, and nonfiber carbohydrate digestibility, and a trend was observed for an increase (P = 0.08) in NDF digestibility with increasing dietary SH addition. Apparent digestibilities of CP and ether extract and ADF digestibility were not affected by SH inclusion rate. Total ruminal concentration of short-chain fatty acids was affected quadratically (P = 0.03) by diet; acetate concentration increased linearly (P < 0.01), whereas quadratic effects were observed on propionate concentration (P = 0.03) and acetate-to-propionate ratio (P < 0.01) with increasing dietary SH. Ruminal pH increased linearly (P < 0.01) as corn was replaced by SH. Nitrogen retention was not affected by the addition of SH, in spite of a linear increase in urinary N (P < 0.01). The replacement of corn grain with up to 45% SH (31.4% of SH in the dietary DM) resulted in a trend to decrease apparent digestibility of DM and to increase NDF digestibility, presenting a fermentation pattern that would reduce the risk of acidosis.

Substitution of crude cell wall for neutral detergent fibre in the equations of the Cornell Net Carbohydrate and Protein System that predict carbohydrate fractions: application to sunflower ( Helianthus annuus L.).

Prediction of carbohydrate fractions using equations from the Cornell Net Carbohydrate and Protein System (CNCPS) is a valuable tool to assess the nutritional value of forages. In this paper, these carbohydrate fractions were predicted using data from three sunflower (Helianthus annuus L.) cultivars, fresh or as silage. The CNCPS equations for fractions B2 and C include measurement of ash and protein-free neutral detergent fibre (NDF) as one of their components. However, NDF lacks pectin and other non-starch polysaccharides that are found in the cell wall (CW) matrix, so this work compared the use of a crude CW preparation instead of NDF in the CNCPS equations. There were no differences in the estimates of fractions B1 and C when CW replaced NDF; however, there were differences in fractions A and B2. Some of the CNCPS equations could be simplified when using CW instead of NDF. Notably, lignin could be expressed as a proportion of DM, rather than on the basis of ash and protein-free NDF, when predicting CNCPS fraction C. The CNCPS fraction B1 (starch + pectin) values were lower than pectin determined through wet chemistry. This finding, along with the results obtained by the substitution of CW for NDF in the CNCPS equations, suggests that pectin was not part of fraction B1 but present in fraction A. We suggest that pectin and other non-starch polysaccharides that are dissolved by the neutral detergent solution be allocated to a specific fraction (B2) and that another fraction (B3) be adopted for the digestible cell wall carbohydrates.