Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.     

The Usefulness of Captive Kept Capercaillie (Tetrao urogallus L.) as the Semen Donors for Artificial Insemination and Gene Pool Preservation In vitro

Captive breeding of birds threatened by extinction in zoological gardens or other closed aviary centres is one of the methods allowing their protection and gene pool preservation ex situ in vivo. Such birds are usually kept in captivity lifelong and serve as parents of several new generations that can be further released into natural environment, or males are used as semen donors for artificial insemination and gene banking. Therefore, the fecundity of such flocks (number of laid egg and spermatozoa quantity and quality) is very important. The aim of this study was to evaluate the usefulness of captive kept capercaillie (Tetrao urogallus L.) as semen donors in three subsequent reproductive seasons, based on the assessment of manually collected semen quality. Male response to dorso‐abdominal massage, ejaculate volume, sperm concentration, motility and morphology were evaluated individually at three succeeding years. Depending on individual male properties and year of collection, the number of positive reactions to semen collection attempts (i.e. ending with ejaculation) varied from 44.4% to 100.0%; single ejaculate volume ranged from 10 to 300 μl, spermatozoa concentration from 10 × 10⁶ per ml to 3520 × 10⁶ per ml and percentage of live morphologically normal spermatozoa from 19.3 to 80.3%. The highest average value (66.7) of semen quality factor (SQF) was noted for a 2‐year‐old male (varying from 1.9 to 258.1), while the lowest for ten‐ (4.8; varying from 0.1 to 17.0) and 7‐year‐old (6.6; varying between 0.6 and 13.6). Assuming that for AI purposes, the ejaculate quality has to be at minimum 10 SQF, obtained results indicate that majority of capercaillie kept in captivity, both young (2–3 years old) and older (up to 10 years old), can be valuable semen producers in succeeding seasons.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.

Methods

Six bovine foetuses were chronic cannulated on the aorta via the medial tarsal artery. Infusion of rabbit anti-bPL IgG was performed during late gestation. Pooled rabbit anti-bPL antisera had a maximal neutralization capacity of 25 μg bPL/mL of immunoglobulin. Interference of rabbit anti-bPL immunoglobulin with radioimmunoassay measurement using guinea pig anti-bPL as primary antibody was first evaluated in vitro. Polyclonal anti-bPL antibodies raised in rabbit were added in foetal sera to produce 100 samples with known antibodies titers (dilutions ranging from 1:2,500 till 1:1,280,000).

Result(s)

Assessment of the interference of rabbit anti-bPL antibody showed that bPL concentrations were significantly lower (P < 0.05) in samples added with dilutions of rabbit antiserum lower than 1:80,000 (one foetus) or 1:10,000 (four foetuses). It was also shown that the recovery of added bPL (12 ng/mL) was markedly reduced in those samples in which exogenous rabbit anti-bPL were added at dilutions lower than 1:20,000. Concentrations of foetal bPL were determined in samples from cannulated foetuses. In foetuses 1 and 6, bPL concentrations remained almost unchanged (<5 ng/mL) during the whole experimental period. In Foetus 3, bPL concentrations decreased immediately after IgG infusion and thereafter, they increased until parturition.

Conclusion(s)

The use of a bPL RIA using a guinea pig anti-bPL as primary antiserum allowed for the measurement of bPL concentrations in foetal plasma in presence of rabbit anti-bPL IgG into the foetal circulation. Long-term foetal catheterization allowed for the study of the influence of direct infusion of anti-bPL IgG on peripheral bPL concentrations in bovine foetuses.     

Osmoregulatory balance in Murray cod, Maccullochella peelii peelii (Mitchell), affected with chronic ulcerative dermatopathy

This study examined the osmoregulatory capability of Murray cod, Maccullochella peelii peelii, affected by chronic ulcerative dermatopathy (CUD) in intensive aquaculture. This condition appears to arise only in facilities utilizing groundwater, with the causative agent suggested to be a water-borne factor. Healthy Murray cod (~ 700 g) were transferred to a CUD-affected farm to monitor the progression of the syndrome and began to show signs of CUD after approximately five months. In order to evaluate possible effects of CUD on osmoregulation; plasma electrolyte concentrations, osmolality, and Na⁺,K⁺-ATPase activities were measured, and gill histology and immunohistochemistry were analyzed. Plasma electrolyte concentrations and osmolality of CUD-affected Murray cod were consistent with reference values determined for non CUD-affected fish. A greater number of gill mucous cells were observed in Murray cod cultured at the CUD-affected farm compared to non CUD-affected fish. We also found an un-identified cell type that was present solely in the gills of CUD-affected Murray cod. Gill Na⁺,K⁺-ATPase activity was significantly higher in severely CUD-affected Murray cod compared to individuals transferred to the CUD-affected farm. While there appeared to be some minor changes in the gills of CUD-affected fish, this study demonstrated that Murray cod were able to effectively osmoregulate, although, perhaps at an energetic cost.     

Oxalate and phytate concentrations in seeds of soybean cultivars [Glycine max (L.) Merr.

This study analyzed soybean seeds from 116 cultivars for total, insoluble, and soluble oxalate (Ox), phytate (InsP6), calcium (Ca), and magnesium (Mg) because of their potential beneficial or harmful effects on human nutrition. These cultivars were divided into four groups (A-D) on the basis of the year and geographic location where they were grown. Oxalate concentration ranged from about 82 to 285 mg/100 g of dry seed. The InsP6 concentration ranged from 0.22 to 2.22 g/100 g of dry seed. There was no correlation between Ox and InsP6 within or among the four groups of cultivars. There was a significant correlation between total Ox and Ca, but not Mg, in group D cultivars (r = 0.3705; p < 0.0005). No significant relationship was found in the group A-C cultivars. Eleven group D cultivars had InsP6 less than 500 mg/100 g, but all had total Ox of 130 mg/100 g or greater. Five cultivars from groups A-C had relatively low InsP6 (group B; < or =1.01 g/100 g) and low Ox (<140 mg/100 g). These cultivars could be useful for producing soy foods beneficial to populations at risk for kidney stones and for improved mineral bioavailability. The Ox and InsP6 concentrations of the cultivars indicate that choosing specific parents could generate seeds in succeeding generations with desirable Ox and InsP6 concentrations.