CD117‐ and vimentin‐positive telocytes in the bovine teat sphincter

Mastitis is a common economically relevant problem in dairy farming. As the major entry for pathogens is the papillary duct, one of the first defence mechanisms is the teat sphincter. This sphincter shows a rhythmic contractility of yet unknown origin. Searching for possible modulatory pacemaker cells, teat sphincters of eight cows were stained immunohistochemically with antibodies against CD117 and vimentin and evaluated microscopically for the presence of telocytes. CD117‐ and vimentin‐positive telocytes with telopodes were found in close contact with smooth muscle cells. Our findings present a first evidence of telocytes in the teat of bovines.     

Effects of in ovo injection of chrysin,quercetin and ascorbic acid on hatchability,somatic attributes,hepatic oxidative status and early post‐hatch performance of broiler chicks

An experiment was conducted to evaluate the effects of in ovo injection of chrysin, quercetin and ascorbic acid on hatchability, somatic attributes, hepatic antioxidant status and early post‐hatch growth performance of broiler chicks. Four hundred and eighty embryonated broiler breeder eggs containing live 18‐day‐old embryos were divided into six groups of 80 eggs each. One group remained intact and served as a control group (i), whereas the other five groups were injected with the prepared injection solutions as follows: (ii) 0.05 ml distilled water; (iii) 0.05 ml distilled water containing 6 mg ascorbic acid; (iv) 0.05 ml dimethyl sulfoxide (DMSO); (v) 0.05 ml DMSO containing 4.5 mg quercetin; and (vi) 0.05 ml DMSO containing 4.5 mg chrysin. The hatchability rate, hatching weight, residual yolk sac weight, yolk sac‐free body weight, liver weight, hepatic glutathione peroxidase and total superoxide dismutase activities, as well as malondialdehyde concentrations, were not affected by the injected solutions. There were no differences between chicks hatched from the control and in ovo injected eggs in weight gain, feed intake and feed conversion ratio from 0 to 11 days of age. However, the specific contrast performed between the in ovo injected groups and intact eggs revealed that in ovo injection significantly increased hatchability rate (p = .0493). This finding also implies that our injection procedure was harmless. In conclusion, the intra‐egg injection of chrysin, quercetin or ascorbic acid at the injection rates used in this study did not have a significant effect on hatchability, somatic characteristics, early growth performance and hepatic antioxidant status of broiler chicks. However, the overall hatchability was higher in the in ovo injected eggs as compared to non‐injected ones. These findings also confirmed the harmlessness of the procedure developed for in ovo injection in this study.     

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.     

Disaccharide combinations and the expression of enolase3 and plasma membrane Ca2+ ATPase isoform in sturgeon sperm cryopreservation

Acipenser sinensis and Acipenser dabryanus are critically endangered species, so germplasm conservation via cryopreservation of sperm is necessary. Disaccharides can act as membrane‐impermeable cryoprotectants, and enolase3 (ENO3) and plasma membrane Ca²⁺ ATPase isoform (PMCA2) are proteins associated with sperm quality. We considered seven characteristics of sperm quality in cultured brood stock from A. sinensis and A. dabryanus. We tested use of sucrose or trehalose alone and in combination at different concentrations for cryopreservation of A. dabryanus sperm. A low concentration of sucrose plus trehalose (S₁₅T₁₅) was optimal. Mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L₁₅T₁₅) gave the best results for both A. sinensis and A. dabryanus. Enolase3 and PMCA2 expression levels were measured in cryopreserved A. sinensis sperm via Western blotting. Relative ENO3 and PMCA2 expression levels were examined, and the relationship between disaccharide composition, sperm quality and protein expression was explored in A. sinensis. The results showed that relative ENO3 and PMCA2 expression levels were the highest at L₁₅T₁₅ in cryopreserved A. sinensis sperm. There were significant positive correlations between ENO3 expression and percentage membrane integrity, and between PMCA2 expression and sperm motility parameters (percentage of motile sperm, curvilinear velocity, straight‐line velocity and average path velocity; p < .05) in cryopreserved A. sinensis sperm. Our results indicate the optimal disaccharide combination and concentrations for cryopreservation of A. sinensis and A. dabryanus sperm and suggest that ENO3 and PMCA2 expression levels could serve as a valuable indicator of sperm quality in A. sinensis.     

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.