Effect of bovine seminal plasma on bovine endometrial epithelial cells in culture

The purpose of this study was to investigate the effect of seminal plasma (SP) from bulls of known fertility on bovine endometrial epithelial cells (bEEC) in culture. The bEEC from passage 5, approximately 5.0–13 × 10⁵ cells per flask, were challenged with SP from bulls of high or low fertility (n = 3 and 2, respectively) or PBS (control), at 1% (75 μl) or 4% (300 μl) and were incubated for 72 hr (n = 13 per challenge). Total cell number and viability of bEEC after challenge with 1% SP from either high‐ or low‐fertility bulls (75H or 75L, respectively) did not differ from controls. In contrast, challenge with 4% of SP from high‐ or low‐fertility bulls (300H or 300L) negatively affected bEEC cell number and viability. Challenge with 300 L had a greater adverse effect than 300H. These results suggest that the negative effect of bovine SP on bEEC is both dose‐dependent and fertility‐dependent.     

Bovine foetal sex determination—Different DNA extraction and amplification approaches for efficient livestock production

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender‐specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell‐free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300–600 bp) in maternal circulation. The aim of this study was to assess this non‐invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non‐pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single‐tube extraction without silicone membranes and phenol–chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real‐time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single‐tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real‐time PCR test sensitivity in Group I was 90% and in Group II 91.6%.     

In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.     

Expression,Purification and Activity Evaluation of Mb1230 Gene of Mycobacterium bovis

To explore the value of Mb1230 protein of Mycobacterium bovis in the diagnosis of bovine tuberculosis,we obtained the Mb1230 gene by PCR and constructed recombinant plasmid pET-22b-Mb1230.Recombinant Mb1230 protein was obtained by IPTG induction and purified by affinity chromatography.The activity of the recombinant protein was evaluated by TST test,IGRA test and indirect ELISA.The size of the recombinant protein matched with the theoretical value proved by SDS-PAGE;Western blotting result showed that the recombinant protein could react with mouse anti-His antibody,and had specific band;The results of TST test,IGRA test and indirect ELISA test also showed the recombinant protein had antigenic activity.The results indicated the recombinant protein Mb1230 had good B cell and T cell activity,so,it had the potential application in the diagnosis of bovine tuberculosis.     

激素对奶牛乳腺上皮细胞乳脂肪合成的调控作用

乳脂肪是高质量的天然脂肪，其可为人类提供营养和能量，在各种膳食脂肪和油类中，是最容易被消化吸收的。乳脂肪是在乳腺中由从头合成或外源摄取的脂肪酸与甘油酯化形成的一种脂类物质，其含量的高低关系着牛奶品质的优劣和乳制品的加工特性。在奶牛的泌乳周期中，乳腺泌乳功能受多种因素影响，其中内分泌腺分泌的多种激素对奶牛乳腺上皮细胞（BMECs）乳脂的合成具有积极的调控作用。综上所述，作者介绍了氢化可的松、催乳素、胰岛素和生长激素4种泌乳相关激素对BMECs乳脂肪合成的调控机理，即从乳脂合成适宜的激素添加量、激素对乳脂球形态的影响方面初步阐释其调控作用，并从乳脂合成的关键酶及转录因子、激素对乳脂合成相关基因表达量方面深入阐释其作用机理，旨在为研究泌乳相关激素对奶牛乳腺内乳脂肪合成的调控机理提供参考。     

Effect of Psammaplin A on in vitro Development of Bovine Aging Oocytes

To investigate the effect of histone deacetylation inhibitor Psammaplin A (PsA) on the development of bovine aging oocytes in vitro,oocytes were randomly divided into control group,aging group and 50 mmol/L PsA treated aging group (PsA group).Immunofluorescence staining and JC-1 were used to detect the blastocyst rate of bovine oocytes after parthenogenetic activation,the number of cells in blastocysts,apoptosis,reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential intensity of embryos.The results showed that the blastocyst rate of the aging group was significantly lower than that of PsA and control groups (P<0.05).The blastocyst rate of PsA group was not significantly different from that of control group (P>0.05).The number of cells in the blastocysts of control group and PsA group were significantly higher than that of aging group (P<0.05).The number of cells in the blastocysts of PsA group was not significantly different from that of control group (P>0.05).The apoptosis rate in aging group was significantly higher than that of control and PsA groups (P<0.05),the apoptosis rate of PsA group was significantly higher than that of control group (P<0.05).The GSH level of MⅡ oocytes in aging group was significantly lower than that of control and PsA groups (P<0.05).There was no significant difference in GSH level between control and PsA groups (P>0.05).The ROS level of the embryos in aging group was significantly higher than that of control and PsA groups (P<0.05).The ROS level in PsA group was significantly higher than that of control group (P<0.05).The mitochondrial membrane potential of early embryos of aging group 4-8 cells was significantly lower than that of control and PsA groups (P<0.05).The mitochondrial membrane potential intensity of control group was significantly higher than that of PsA group (P<0.05).In summary,PsA could effectively delay the aging of bovine oocytes and improve the quality of oocytes.