Differences in maternal plane of nutrition and body condition during late gestation coupled with estrus synchronization at weaning do not result in differences in embryonic development at 4 days of gestation.

This study was conducted to assess the effects of dietary energy in late pregnancy and hormone therapy at weaning on plasma metabolite profile, litter performance, reproductive parameters, and embryo viability in the second pregnancy. A total of 23 first-parity sows at 75 d of pregnancy were randomly allocated to 4 treatments. Treatments were factorial (2 × 2) combinations of 2 nutritional strategies [standard-energy feed (SEF) and high-energy feed (HEF)] and 2 hormone therapies [600 IU eCG and 2.5 mg swine LH 72 h later (HO) and no hormone (WH)]. Sows were weighed weekly from 75 d of pregnancy until 3 d before farrowing; 1 d after farrowing; 7, 14, and 21 d into lactation; and at weaning. Back fat (BF) was measured at 75 d of pregnancy, 3 d before farrowing, and at weaning. Average daily gain and ADFI were also calculated. Plasma metabolites were analyzed after 82, 89, 96, and 103 d of pregnancy, at farrowing, and after 7, 14, and 21 d of lactation. Embryo viability was assessed after 4.55 d of second pregnancy. During pregnancy, HEF-treated sows displayed greater BW (P < 0.05) compared with SEF-treated females, but no differences were observed during lactation. There were no differences in BW of the piglets caused by the treatments. High-energy-treated females showed superior BF (P > 0.05) in all periods; however, significant differences were detected only at the prefarrowing measurement (P < 0.05). No differences in ADFI were observed during lactation. The SEF group showed positive ADG, whereas the HEF group showed negative ADG (0.216 vs. -0.266 kg/d for SEF and HEF, respectively; P < 0.05). High-energy-treated sows presented greater concentrations of total cholesterol after 89 and 103 d of pregnancy and greater concentrations of high-density lipid cholesterol (HDL) after 89 and 96 d. At farrowing and 14 and 21 d of lactation, NEFA concentrations were greater (P < 0.05) in the HEF group. After hormone treatment, no differences were observed on weaning-to-estrus intervals and estrus duration. Greater mobilization of body reserves observed in the HEF group during lactation did not affect reproductive performance negatively, suggesting that metabolic status was adequate for the first lactational catabolism.

α-6 integrin expression in bovine spermatogonial cells purified by discontinuous Percoll density gradient.

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 × 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α-6 integrin expression. Flow cytometry analysis demonstrated no differences in the α-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to α-6 integrin, the expression of GFRa-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α-6 integrin expression.

Effect of low oxygen tension atmosphere and maturation media supplementation on nuclear maturation, cortical granules migration and sperm penetration in swine in vitro fertilization.

The aim of this study was to evaluate the efficiency of low oxygen tension (5% CO(2) , 5% O(2) and 90% N(2) ) on in vitro oocyte maturation using defined media (0.1% polyvinyl alcohol - PVA) or 10% porcine follicular fluid (PFF)-supplemented media. To achieve this goal, oocytes were evaluated regarding cortical granules (GCs) migration, nuclear maturation and sperm penetration. Oocytes were in vitro matured under different conditions: 5% or 20% O(2) atmosphere and 0.1% PVA- or 10% PFF-supplemented media and evaluated at 0 and 44 h of maturation. To evaluate the migration of CGs and nuclear maturation, by confocal microscopy, oocytes were incubated with 100 µg of FITC-PNA/ml and 10 µg/ml of propidium iodide. To address sperm penetration, after maturation, in vitro fertilization for 6 h and in vitro culture for 18 h, zygotes were incubated with 10 mg/ml Hoechst 33342. Pronuclei and polar bodies were quantified using an epifluorescence microscope. Atmosphere conditions did not affect the CGs migration, but media supplementation did. Oocytes matured in 10% PFF media had a higher percentage of CGs in the oocyte periphery than oocytes matured in PVA-supplemented media. However, this fact did not have effect on in vitro sperm penetration levels. No effect of atmosphere conditions and media supplementation was observed on the rates of metaphase II oocytes. Therefore, the use of low oxygen tension in association with PVA maturation media does not improve the in vitro maturation system of porcine oocytes, because its use did not improve nuclear maturation, CGs migration and zygotes monospermic rates.

Serum starvation and full confluency for cell cycle synchronization of domestic cat (felis catus) foetal fibroblasts.

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < or = 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < or = 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.

Effects of ethanol on synthesis of prostaglandin F2α in bovine females.

Ethanol stimulates the production of prostaglandins in many species. The purpose of this study was to verify the effect of ethanol on the production of prostaglandin F2α (PGF2α) and luteolysis in bovine females. In the first experiment, Holstein cows at day 17 of the oestrous cycle were treated with 100% ethanol (0.05 ml/kg of body weight, IV; n = 5), saline (0.05 ml/kg of body weight, IV; n = 4) or synthetic prostaglandin (150 µg of D-cloprostenol/cow, IM; n = 4). The plasma concentrations of 13, 14-dihydro-15-keto PGF2α (PGFM; the main metabolite of PGF2α measured in the peripheral blood) were assessed by radioimmunoassay (RIA). There was an acute release of PGFM in response to ethanol comparing to other treatments (p ≤ 0.05). However, only cows treated with PGF2α underwent luteolysis. In the second experiment, endometrial explants of cross-bred beef cows (n = 4) slaughtered at day 17 of the oestrous cycle were cultured for 4 h. During the last 3 h, the explants were cultured with medium supplemented with 0, 0.1, 1, 10 or 100 µl of 100% ethanol/ml. Medium samples were collected at hours 1 and 4 and concentrations of PGF2α were measured by RIA. Ethanol did not induce PGF2α production by the endometrium. In conclusion, ethanol does not cause luteolysis in cows because it stimulates production of PGF2α in extra-endometrial tissues.

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency.

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A3 (CMA3) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA3 staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA3-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA3-positive sperm cells compared to the others. CMA3 is a simple and useful tool for detecting sperm protamine deficiency in bulls.

Effects of serum deprivation and cycloheximide on cell cycle of low and high passage porcine fetal fibroblasts.

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 +/- 0.65) when compared with non-starved cells (71.44 +/- 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.