[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Rosmarinic acid improves boar sperm quality, antioxidant capacity and energy metabolism at 17°C via AMPK activation.

Boar sperm are susceptible to oxidative damage caused by reactive oxygen species (ROS) during storage. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important therapeutic target, because it is a cellular metabolism energy sensor and key signalling kinase in spermatozoa. We evaluated the effects of rosmarinic acid (RA), an antioxidant, on boar sperm during liquid storage to determine whether it protects boar sperm via AMPK activation. Boar ejaculates were diluted with Modena extender with different concentrations of RA and stored at 17°C for 9 days. Sperm quality parameters, antioxidant capacity, energy metabolism, AMPK phosphorylation and fertility were analysed. Compared with the control, 40 µmol/L significantly improved sperm motility, plasma membrane integrity and acrosome integrity (p < .05). The effective storage time of boar sperm was up to 9 days. On the third and seventh days, the sperm with RA exhibited increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, adenosine triphosphate (ATP) content, mitochondrial membrane potential (ΔΨm) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, whereas malondialdehyde (MDA) content was significantly decreased (p < .05). Western blot showed that RA, as well as AICAR (AMPK activator), promoted AMPK phosphorylation, whereas Compound C (AMPK inhibitor) inhibited this effect. The sperm-zona pellucida binding experiment showed that 40 µmol/L RA increased the number of sperm attached to the zona pellucida (p < .05). These findings suggest meaningful methods for improved preservation of boar sperm in vitro and provide new insights into the mechanism by which RA protects sperm cells from oxidative damage via AMPK activation.

Effect of addition of melatonin on liquid storage of ram semen at 4°C.

Adding a certain amount of antioxidants to semen extender has been shown to improve semen quality. The aim of present study was to elucidate whether the supplementation of melatonin to the Tris-based extender (CTR) could enhance the quality of ram spermatozoa during storage at 4°C. Ram semen samples were collected and diluted with CTR extender containing different concentrations (0, 0.05 (M 0.05), 0.1 (M 0.1), 0.2 (M 0.2) or 0.4 (M 0.4) mM) of melatonin. Sperm routine indicators, mitochondrial activity, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content were analysed in control and melatonin treatment groups. The higher per cent of motility, plasma membrane integrity, mitochondrial activity and T-AOC activity was observed in M 0.05, M 0.1 and M 0.2 groups compared to control group at 5 days of storage (p < 0.05), while lower percentage of MDA content was observed among these groups (p < 0.05). In addition, there were no significant differences in acrosome integrity among the control and M 0.05, M 0.1 and M 0.2 groups during the experiment. The above results show that the addition of 0.05, 0.1, 0.2 mM of melatonin is beneficial to the preservation of ram semen during liquid storage at 4°C mainly through antioxidative stress.

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.

Association of heat shock protein 90 with motility of post-thawed sperm in bulls.

The correlation between the 90 kDa heat-shock protein (HSP90) and sperm quality following the process of freezing-thawing in bulls has not been studied clearly. Therefore, the objective of the present was to clarify the relationship between HSP90 level and semen parameters during the process of cryopreservation in bulls. Semen samples from 5 Holstein bulls were obtained by artificial vagina. Characteristics of these semen at three stages (fresh, after equilibration and frozen-thawed), including motility, plasma membrane integrity and acrosome integrity were evaluated. The mRNA expression level of HSP90 at the three stages was evaluated by using quantitative Real-Time PCR. Meanwhile, the protein level of HSP90 expression at the three stages was detected according to Western blot. The results showed that sperm parameters evaluated in fresh semen was the highest in the three groups. Sperm parameters in semen after equilibration were lower than those in fresh semen (P>0.05) and higher than those in post-thawed semen (P<0.05). Sperm parameters in frozen-thawed semen were the lowest among the three groups (P<0.05). This study indicated that HSP90 expression is proportional to sperm quality. HSP90 expression level in fresh semen was significantly higher than that in frozen-thawed semen (P<0.05). Although no significant differences in HSP90 expression were observed between fresh semen and semen after equilibration (P>0.05). Results in this study suggest that HSP90 level in bull spermatozoa was gradually declined following the process of freezing-thawing, and might be associated with sperm motility, plasma membrane integrity and acrosome integrity.

Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue.

Trehalose is widely used for cryopreservation of various cells and tissues. Until now, the effect of trehalose supplementation on cell viability and antioxidant enzyme activity in frozen-thawed bovine calf testicular tissue remains unexplored. The objective of the present study was to compare the effect of varying doses of trehalose in cryomedia on cell viability and key antioxidant enzymes activities in frozen-thawed bovine calf testicular tissue. Bovine calf testicular tissue samples were collected and cryopreserved in the cryomedias containing varying doses (0, 5, 10, 15, 20 and 25%; v/v) of trehalose, respectively. Cell viability, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) content and malondialdehyde (MDA) content were measured and analyzed. The results showed that cell viability, T-AOC activity, SOD activity, CAT activity and GSH content of frozen-thawed bovine calf testicular tissue was decreased compared with that of fresh group (P<0.05). MDA content in frozen-thawed bovine calf testicular tissue was significantly increased compared with that of fresh group (P<0.05). The cryomedia added 15% trehalose exhibited the greatest percentage of cell viability and antioxidant enzyme activity (SOD and CAT) among frozen-thawed groups (P<0.05). Meanwhile, GSH content was the lowest among frozen-thawed groups (P<0.05). However, there were no significance differences in MDA content among the groups added 10, 15 and 20% trehalose (P>0.05). In conclusion, the cryomedia added 15% trehalose reduced the oxidative stress and improved the cryoprotective effect of bovine calf testicular tissue. Further studies are required to obtain more concrete results on the determination of antioxidant capacity of trehalose in frozen-thawed bovine calf testicular tissue.

Effects of testicular interstitial fluid on the proliferation of the mouse spermatogonial stem cells in vitro.

Spermatogenesis is a process in adult male mammals supported by spermatogonial stem cells (SSCs). The cultivation of SSCs has potential value, for example for the treatment of male infertility or spermatogonial transplantation. Testicular interstitial fluid was added to culture medium to a final concentration of 5, 10, 20, 30 or 40%, in order to investigate its effects on proliferation of mouse SSCs in vitro, Alkaline phosphatase (AKP) assay, reverse transcription polymerase chain reaction (RT-PCR) analysis and indirect immunofluorescence of cells were performed to identify SSCs, and the proliferation rate and diameters of the SSCs colonies were measured. The results showed that the optimal addition of testicular interstitial fluid to culture medium was 30%. When medium supplemented with 30% testicular interstitial fluid was used to culture mouse SSCs, the optimum proliferation rate and diameter of the cell colonies were 72.53% and 249 µm, respectively, after 8 days in culture, values that were significant higher than those found for other groups (P < 0.05). In conclusion, proliferation of mouse SSCs could be promoted significantly by supplementation of the culture medium with 30% testicular interstitial fluid. More research is needed to evaluate and understand the precise physiological role of testicular interstitial fluid during cultivation of SSCs.

HSP90 expression correlation with the freezing resistance of bull sperm.

To date, there has been little improvement in cryopreservation of bull sperm due to lack of understanding of the freezing mechanisms. Therefore, this study set out to investigate expression levels of fertility-associated proteins in bull sperm, and in particular the relationship between the 90 kDa heat-shock protein (HSP90) and the sperm characteristics after freezing-thawing. Semen was collected from eight Holstein bulls by artificial vagina. Characteristics of these fresh semen, including sperm motility, morphology, viability and concentration, were evaluated. Sperm quality was also assessed after freezing-thawing. Eight ejaculates were divided into two groups based on freezing resistance and sperm motility. Sperm proteins were extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting were performed. SDS-PAGE results showed that there was substantial diversity in 90 kDa proteins in the frozen-thawed sperm and HSP90 was confirmed as one of the 90 kDa proteins by western blot. This study indicated that HSP90 expression correlated positively with sperm quality. The amount of expressed 90 kDa proteins in the high freezing resistance (HFR) group was significantly higher than that in the low freezing resistance (LFR) group (P < 0.05). Thus, higher expression of HSP90 could probably lead to the higher motility and freezing resistance of sperm found after freezing-thawing. Therefore, we concluded that level of HSP90 expression could be used to predict reliably and simply the freezing resistance of bull sperm.

Effects of low-density lipoproteins extracted from different avian yolks on boar spermatozoa quality following freezing-thawing.

Low-density lipoproteins (LDL) is known to protect boar sperm during freezing-thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing-thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen-thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen-thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen-thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.

Cryoprotective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells.

Spermatogonial stem cells (SSCs) have the ability to self-renew and offer a pathway for genetic engineering of the male germ line. Cryopreservation of SSCs has potential value for the treatment of male infertility, spermatogonial transplantation, and so on. In order to investigate the cryopreservation effects of different cryoprotectants on murine SSCs, 0.2 M of low-density lipoproteins (LDL), trehalose and soybean lecithin were added to the cryoprotective medium, respectively, and the murine SSCs were frozen at -80°C or -196°C. The results indicated that the optimal recovery rates of murine SSCs in the cryoprotective medium supplemented with LDL, trehalose and soybean lecithin were 92.53, 76.35 and 75.48% at -80°C, respectively. Compared with freezing at -196°C, the optimum temperature for improvement of recovery rates of frozen murine SSCs, cryopreservation in three different cryoprotectants at -80°C, were 17.11, 6.68 and 10.44% respectively. The recovery rates of murine SSCs in the cryoprotective medium supplemented with 0.2 M LDL were significantly higher than that of other cryoprotectants (P < 0.05). Moreover, the recovery rates were demonstrated to be greater at -80°C compared with at -196°C (P < 0.05). In conclusion, 0.2 M of LDL could significantly protect murine SSCs at -80°C. In the freezing-thawing process, LDL is responsible for the cryopreservation of murine SSCs because it can form a protective film at the surface of membranes. However, more research is needed to evaluate and understand the precise role of LDL during the freezing-thawing of SSCs.

Metformin improves sheep sperm cryopreservation via vitalizing the AMPK pathway.

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is a key regulator of sperm function and physiological metabolism. Metformin, an inexpensive and effective antioxidant, is known to play an important role in the activation of AMPK. Therefore metformin has potential to improve sperm cryopreservation. The aim of this study was to investigate the effect of metformin during semen cryopreservation of sheep and to find the most effective concentration in freezing extender. Semen were cryopreserved with extender containing different concentrations of metformin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L). Sperm motility, acrosome integrity and plasma membrane integrity were measured after semen freezing and thawing. All results showed that sperm quality was significantly increased in the 1.0 mmol/L metformin-treated group compared with the control group (P < 0.05). In addition, the study showed that metformin effectively reduced the content of malondialdehyde (MDA) and reactive oxygen species (ROS), and increased the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) of freeze-thawed sperm (P < 0.05). The optimal concentration of metformin was 1.0 mmol/L. Moreover, the results showed that AMPK was localized in the acrosome region, junction and midsection of sperm, and p-AMPK was distributed in the post-acrosomal region, junction and midsection. Western blot analysis indicated that 1.0 mmol/L metformin stimulated the phosphorylation of AMPK in sperm. Further results showed that 1.0 mmol/L metformin significantly increased the mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake and lactate efflux of post-thawed sperm through the AMPK pathway, improved sperm quality, and increased the cleavage rate of in vitro fertilization (P < 0.05).

Effect of Astragalus polysaccharides on the cryopreservation of goat semen.

During cryopreservation, sperm encounters oxidative stress induced by excessive reactive oxygen species (ROS), destroying the sperm plasma membrane structure and reducing its physiological functions. The present study aimed to evaluate the effect of Astragalus polysaccharides (APS) on the cryopreservation of dairy goat semen. Semen was collected from six goats, and then qualified semen with movement >80% was selected after preliminary evaluation. The semen was divided into six aliquots, diluted with dairy goat semen extender (1:10) at 37 °C, containing 0 g/L (control), 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L and 0.5 g/L APS, cryopreserved, and stored in liquid nitrogen (-196 °C). Sperm quality was assessed after freeze-thawing. The highest sperm motility, motion performance, plasma membrane integrity, acrosome integrity, and antioxidant properties (total antioxidant capacity and levels of antioxidant enzymes) were recorded (P < 0.05) in the 0.2 g/L APS group after the semen was freeze-thawed. The control and the optimal group (0.2 g/L) were then selected to analyze the effects of APS on sperm energy metabolism (mitochondrial membrane potential [MMP] and adenosine triphosphate [ATP]), sperm apoptosis, and the expression of the AMPK signaling pathway. The results showed that treatment with 0.2 g/L APS increased sperm MMP and ATP content after freeze-thawing, reduced sperm apoptosis by regulating apoptosis-related proteins, and promoted AMPK phosphorylation by activating the AMPK signaling pathway. The cleavage rate of frozen goat sperm during in vitro fertilization (IVF) was also observed to increase. These findings suggest meaningful ways to improve cryopreservation of dairy goat semen and provide new insights into the mechanism by which APS protects sperm from oxidative damage via AMPK activation.

Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells.

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

The advantages of low-density lipoproteins in the cryopreservation of bull semen.

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen-thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk (P<0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups (P>0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) (P<0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.

Melatonin Protects Goat Spermatogonial Stem Cells against Oxidative Damage during Cryopreservation by Improving Antioxidant Capacity and Inhibiting Mitochondrial Apoptosis Pathway.

Spermatogonial stem cells (SSCs) are the only adult stem cells that pass genes to the next generation and can be used in assisted reproductive technology and stem cell therapy. SSC cryopreservation is an important method for the preservation of immature male fertility. However, freezing increases the production of intracellular reactive oxygen species (ROS) and causes oxidative damage to SSCs. The aim of this study was to investigate the effect of melatonin on goat SSCs during cryopreservation and to explore its protective mechanism. We obtained SSCs from dairy goat testes by two-step enzymatic digestion and differential plating. The SSCs were cryopreserved with freezing media containing different melatonin concentrations. The results showed that 10-6 M of melatonin increased significantly the viability, total antioxidant capacity (T-AOC), and mitochondrial membrane potential of frozen-thawed SSCs, while it reduced significantly the ROS level and malondialdehyde (MDA) content (P < 0.05). Further analysis was performed by western blotting, flow cytometry, and transmission electron microscopy (TEM). Melatonin improved significantly the enzyme activity and protein expression of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (P < 0.05), thereby activating the antioxidant defense system of SSCs. Furthermore, melatonin inhibited significantly the expression of proapoptotic protein (Bax) and increased the expression of antiapoptotic proteins (Bcl-2 and Bcl-XL) (P < 0.05). The mitochondrial apoptosis pathway analysis showed that the addition of melatonin reduced significantly the mitochondrial swelling and vacuolation, and inhibited the release of cytochrome C from mitochondria into the cytoplasm, thereby preventing the activation of caspase-3 (P < 0.05) and inhibiting SSC apoptosis. In addition, melatonin reduced significantly the autophagosome formation and regulated the expression of autophagy-related proteins (LC3-I, LC3-II, P62, Beclin1, and ATG7) (P < 0.05), thereby reversing the freeze-induced excessive autophagy. In summary, melatonin protected goat SSCs during cryopreservation via antioxidant, antiapoptotic, and autophagic regulation.

Epidemiological big data and analytical tools applied in the control programmes on parasitic diseases in China: NIPD's sustained contributions in 70 years.

The analysis of epidemiological data has played an important role for the academic research carried out by the National Institute of Parasitic Diseases, China CDC, since its foundation in 1950s. Those researches, e.g., the temporal-spatial patterns of disease transmission and the identification of risk factors, have contributed significantly to the national parasitic disease control and elimination programmes in China. With the development and application of epidemiological data analysis in the last decade, all research results improve our understanding of parasitic diseases epidemiology and related health issues through the application platform of epidemiological big data and analytical tools. In particular, implementation research on analytical predictions on disease outbreak or epidemic risks have provided references to the scientific guidance on effective preventions and interventions in the parasitic disease elimination in China, such as fliariasis, malaria and schistosomiasis. This review has reflected the function of data accumulation and application of temporospatial tools in parasitic diseases control, and the ways of the NIPD's sustained contributions to the disease control programmes in China.

Recombinant human antithrombin expressed in the milk of non-transgenic goats exhibits high efficiency on rat DIC model.

Plasma-derived antithrombin (pAT) is often used for the treatments of disseminated intravascular coagulation (DIC) patients. In this paper, the recombinant adenovirus vector encoding human antithrombin (AT) cDNA was constructed and directly infused into the mammary gland of two goats. The recombinant human antithrombin (rhAT) was purified by heparin affinity chromatography from the goat milk, and then used in the treatment of thirty lipopolysaccharide (LPS) induced DIC rats. A high expression level of rhAT up to 2.8 g/l was obtained in the milk of goats. After purification, the recovery rate and the purity of the rhAT were up to 54.7 +/- 3.2% and 96.2 +/- 2.7%, respectively. In blood of the DIC rat model treated with rhAT, the levels of antithrombin and thrombin-antithrombin (TAT) were augmented significantly; meanwhile the consumption of fibrinogen and platelet was reduced significantly, and the increase of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentration was restrained modest and non-significant. For the above DIC indexes, there were no differences between pAT and rhAT (P > 0.05). Our results demonstrated that the way we established is a pragmatic tool for large-scale production of rhAT, and the rhAT produced with this method has potential as a substitute for pAT in the therapy of DIC patients.

Effect of Gynostemma Pentaphyllum polysaccharide on boar spermatozoa quality following freezing-thawing.

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen-thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P<0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P<0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P<0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P>0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P>0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen-thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.

Effects of sulfanilamide on boar sperm quality, bacterial composition, and fertility during liquid storage at 17°C.

Sulfanilamide (SA) is an effective broad-spectrum antibacterial agent in human and veterinary medicine. The purpose of this study was to evaluate the effects of SA on boar sperm quality during liquid storage at 17°C and determine the optimal concentration of SA and its effects on bacterial growth, microbial composition, and maternal fertility. Boar ejaculates were diluted with a basic extender, containing different concentrations of SA, and stored in a 17°C incubator for 6 days. The sperm motility, plasma membrane integrity, and acrosome integrity were measured daily. The results showed that when the concentration of SA was 0.02 g/L, the sperm quality parameters were significantly higher than those of all other treatment groups (p < .05). We also monitored the bacterial growth and compared the differences in the microbial species between the 0.02 g/L SA group and the control by 16S rDNA sequencing. The results revealed that some bacteria, such as Staphylococcus and Pseudomonas, were considerably lower in the 0.02 g/L SA group than in the control group (p < .05). In addition, preserved semen was used for artificial insemination, and results showed that 0.02 g/L SA group had a higher litter size, and its pregnancy rate was 92.5%.

Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing.

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing-thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P<0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100mM trehalose, but cryopreservation could increase the degree of DNA damage (P<0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.