Alpase of Follicles during the Period before and after Ovulation in Superovulated Rabbits

The Alpase of follicles was studied systematically during the period before and after ovulation in superovulated rabbits.The locations and changes of follicular Alpase varied with the four kinds of physiological status before and after ovulation.Alpase activity was the highest at oestrus and started to drop at the beginning of ovulation,then it increased 15h later and decreased slightly 39h afterwards the beginning of ovulation.Alpase was found on the follicular basement membrane at ocstrus,The synthesis of fiber in fiberblasts was conncted with Alpase activity.     

Study on features of female fetal reproductive system development in Chinese northeast fine-wool sheep

Some charaterasics of female fetal reproductive system were studied in different gestation phases of Chinese northeast fine-wool sheep.The results showed that reproductive system consisted of ovary,oviduct,uterus and genitalia.The fetal ovaries were granular in early phase and became elliptic in later phase.The functional formula of ovarian weight,length and volume was obtained. Primary oocyte was encapsulated by monolayer cells in 7-week fetal ovary cortex.Primordial follicles were formed in 8-week fetal ovary,the follicular cells were sporadically arranged and not always regular.Complete organization of primordial follicles appeared until 10-week gestation,most of them scattered in nests in the following weeks of gestation.Two types of follicular complexes were found in fetal.There were continuous and quick mitosis of oogonia in fetal ovaries,large complexes were formed by oogoniaes encapsulated by multilayer of follicular cells,then they differentiated into small complexes,one or more primary oocytes were formed by mitosis of oogonia,primordial follicles which were caused by follicular cell approaching into them developed.     

Artificial Induction of Twinning by an Active Immunization of Beef Cows Against Inhibin Partially Purified from Porcine Seminal Plasma

Two hundred and seventy multiparous Chinese Yellow cattle (beef) were selected at 1 to 3months postpartum and divided into three groups (90 cows for each). Animals were given both a primary and booster immunizations with a total dose of 3 mg (Group Th) or 1.5 mg (Group Tl) of seminal preparation containing inhibin activity, emulsified with Freund‘s complete adjuvant and incomplete adjuvant (for booster), at 3 or 4-week intervals. Other cows were treated with the same volume of seminal preparation without inhibin activity as procedures mentioned above to serve as a control (Group C). Artificial inseminations were given twice at 8 - 12 h intervals when the cow was in heat. Jugular venous blood samples were collected from each cow and used to assay the presence of antibody against seminal preparation by double-diffusion in agar precipitation test and to detect the titer of inhibin antibody by an ELISA method. Data from 247 cows showed that 83.9% (73/87) of cows were in estrus and ovulated 89 ova altogether, of which 19 cows ovulated twin ova and 15 cows produced twins in Group Th (n = 87). However, only 61.1% (44/72) of cows in Group Tl (n=72) and 62.5% (55/88) of cows in Group C were in estrus and ovulated 46 and 52 ova altogether respectively.The ovulation rate (1.27 + 0.03), calving rate ( 126.3% ) and twinning rate (26.3%) in Group Th were greater than those in Groups Tl or C (P＜0.01). Furthermore, the ovulation rate was associated with antibody titer in sera of immunized animals (r = 0.7507, P ＜0.01). These results indicate that active immunization of postpartum cows against inhibin purified from porcine seminal plasma may increase the ovulation rate and induce twinning, suggesting the potential to develop a method to improve fertility in cows.     

中国农业科学

In this experiment, the bovine follicular oocytes were aspirated from the ovaries of Chinese Holsteins with laparoscope made in China. The results were as following: for identifying the suitable negative aspiration pressure, six different pressures (50, 100, 150, 200, 250 and 300mmHg)were tested. The aspiration pressure of 100mmHg was the best. Its oocyte recovery rate was 37. 2%, and G I , G Ⅱ oocyte rate was 89. 5%. The heifers were picked up by laparoscope once or twice a week. Each heifer was collected with 2. 4 oocytes once a week or 4. 4 oocytes twice a week.Its oocyte recovery rate was 48. 0% and the G Ⅰ ,G Ⅱ oocyte rate was 93. 5%. In addition, 1.9 oocytes were collected from each cow once a week or 5.4 oocytes from each cow twice a week. Its oocyte recovery rate was 51.7% and the G Ⅰ , G Ⅱ oocyte rate was 85. 1%. It showed that it was possible to pick up bovine oocyte twice a week. Two cows were picked up twice a week for several weeks(53 times). 268 follicles were aspirated(5.1 follicles per cow per time), and 141 oocytes were recovered(2.7 oocytes per cow per time). The oocyte recovery rate was 52.5%, and the G Ⅰ , G Ⅱ oocyte rate was 85. 1%. It was advisable to pick up oocytes twice a week continuously. Some cows in estrous cycles were superovulated with PMSG(500IU). Each of them could be recovered 2.3 follicles and 1.1 oocytes, the others were superovulated with FSH(0. 7mg) , each of them could be aspirated with 4.4 follicles and 2.3 oocytes. It was obvious that the effect of OPU(oocyte pick up) by superovulation with FSH was much better than that with PMSG. The best time for OPU with laparoscope was at the beginning of cow's estrous cycles. At the first day of their estrus, each of them could be averagely aspirated with 8 follicles and 5.7 oocytes.     

Morphological and Hormonal Identification of Porcine Atretic Follicles and Relationship Analysis of Hormone Receptor Levels During Granulosa Cell Apoptosis In vivo

Recent reports have demonstrated that follicular atresia is initiated or caused by granulosa cell apoptosis followed by theca cell degeneration in mammalian ovaries, but the mechanism of follicular atresia is still to be elucidated. Therefore, our present study was designed to examine our hypothesis that the changes of follicular microenvironment induce the granulosa cell apoptosis during pocrine follicular atresia in vivo. We firstly isolated intact porcine antral follicles and identified them into three groups, healthy follicles （HF）, early atretic follicles （EAF） and progressed atretic follicles （PAF） through morphology and histology. To further confirm their status, we detected hormone levels in follicular fluids and the expression level of apoptosis gene Bax in granulosa cells. The rate of progesterone （P） and estradiol （E2） was increased with the expression of Bax, indicating hormone can be used as a marker of granulosa cell apoptosis or follicular atresia. Finally, we analyzed the expression level of hormone receptor genes in granulosa cells and their relationship with follicular atresia. In PAF, the expression of Progesterone receptor （PGR） was increased significantly while estradiol receptor （ER） had no notable changes, which suggesting the increased-PGR accelerated the effect of P-stimulated granulosa cell apoptosis. The dramatic increasing of androgen receptor （AR） expression in PAF and the obvious increase of tumor necrosis factor-u receptor （TNFR） in EAF indicated that there are different pathways regulating granulosa cell apoptosis during follicular atresia. Together, our results suggested that different pathways of granulosa cell apoptosis was induced by changing the follicular microenvironment during follicular atresia.     

Relationship Between the mRNA Expression Level of TGF-β Receptor Genes in Tissues and Ovulation Rate in Hu Sheep

Eight ewes of Hu sheep which bred multi-lamb were used as the high-fecundity group and the other eight ewes of Hu sheep which bred single lamb were used as the control group to investigate the relationship between the mRNA expression level of TGF-β receptor genes in tissues and ovulation rate in Hu sheep. Cloprostenol sodium was injected to make the synchronization of estrus treatment, then all ewes were slaughtered within 24-36 h after empathema and the ovaries were collected. Furthermore, the number of ovulation points was counted to determine ovulation rate for each sheep. Tissue expression analysis was conducted by RT-PCR for one ewe form the high-fecundity group and the relationship between the mRNA expression of genes encoding TGF-β receptors and ovulation rate was detected by real-time fluorescent quantitative PCR. The results showed that the relative expression level of TGF-flR I gene in the reproductive organ was significantly higher than in the lung and muscle （P 〈 0.01）, while relative expression level of TGF-βR H in reproductive organ was significantly higher than that of other tissues （P 〈 0.01）, indicating that these are highly expressed genes in the ovary. In addition, mRNA expression level of TGF-βR I and TGF-flRH in the ovaries of the high-fecundity group were significantly higher （P〈 0.01） and obviously higher （P= 0.011） than the control group, respectively. The mRNA expression level of TGF-βR I and TGF-βR H had a positive correlation with ovulation rate and the correlation coefficients were 0.562 （P〉 0.05） and 0.711 （P〈 0.05）, respectively. It is suggested that TGF-β receptors have close relationship with highfecundity in Hu sheep.     

Male Sterile Lines of Zinnia elegans and Their Cytological Observations

In order to find out a new pathway for utilizing heterosis of Zinnia elegans and accelerate breeding process, the mechanism of anther development of a male sterile line was explored. Backcross, sibmating, selling of fertile plants and testcross with inbred lines were analyzed and identified in the field, and cytology was observed. Recessive nucleus male sterile line AH209AB capable of being a maintainer was obtained by successive backcrosses with male sterile plants and fertile F1 plants as male parents. Cytological and anatomical studies indicated that： （1） The wall of normal anther was constituted of four layers of cells such as epidermis, powder chamber wall, middle level and tapetum cells. The process in meiosis of pollen mother cell in Zinnia elegans was normal and cytoplasm divided simultanously. Mature pollen grain was tricellular type. （2） The petal of male sterile plant degraded as a thread-like structure, the stamens were villiform in appearance and no pollens were formed. The result showed that the anther of male sterile plant no longer proceed to differentiate spore mother cell and the pollen sac after the formation of the tissue of sporogenous cells, there was no evident boundary between tapetum cell, middle lamella and inner wall of PMC, tapetal cells did not develop from the very beginning. So the abortion type was completely structural male sterility. The male sterile line belongs to non-sporange male sterile type and is of great use in F1 seeds production.     

Effect of Rapid Cold Hardening on the Cold Tolerance of the Larvae of the Rice Stem Borer,Chilo suppressalis （Walker）

The effects of rapid cold hardening （RCH） on the cold tolerance of the last instar larvae of Chilo suppressalis （Walker） were evaluated for the first time. The discriminating temperature, induction, detection, duration and extent of RCH of the larvae in the laboratory were tested, and the supercooling points （SCPs） and the contents of water and lipid of the larvae after RCH treatment were determined, respectively. The results showed that the discriminating temperature of the larvae was about -21℃. Mean survival rates of the larvae which exposed to either 0 or 5℃ for 2 and 4 h before exposure to the discriminating temperature for 2 h were significantly higher than those of the control groups （P 〈 0.05）. Moreover, the highest survival rate appeared in the larvae after 0℃ for 4 h treatment. The protection against low temperature gained by RCH at 0℃ for 4 h was rapidly lost on return to 28℃. Mean survival rates of RCH larvae were significantly higher than those of non-acclimated （NACC） larvae and acclimation （ACC） larvae when they were exposed to the discriminating temperature for 2 or 4 h （P〈 0.05）. Moreover, the rates of NACC, ACC, RCH and ACC ＋ RCH larvae from 2 to 6 h to the discriminating temperature resulted in a significant decline. The values of SCPs and the contents of lipid of the larvae which exposed to either 0 or 5℃ for 2 and 4 h showed no significant difference at 0.05 level compared to those of the control groups. But the contents of water in the larvae were obviously decreased. Therefore, it could be concluded that RCH could enhance cold tolerance and affect partly physiological and biochemical components of the larvae of C. suppressalis, but the underlying mechanisms needs to be further explored.     

Histological Study on the Polyovular Folliclular Development of Chinese Prematural Lubei White Goat

The present study was conducted to investigate the histological characteristics of Polyovular follicles in Chinese Lubei white female goats with age 2-5 months. The results suggested that the healthy antral follicles with twin-oocyte could be observed. There were many multi-oocyte phenomena in primordial and primary follicles with a proportion of nearly 75% in primordial follicles. Primordial follicle was in the outer of cortex in ovaries and most of them remained active. The nest of multi-nuclear primordial follicle was developing into primordial follicle with 1 to 3 nuclei.     

Comparison of Different Modes of Molding in Canine Sepsis

Sepsis can cause a series of damages to various organs of the body, so it has always been regarded as a hot research topic in veterinary clinic.Aiming at the present situation of high morbidity and mortality of canine sepsis, in order to further explore the pathogenesis of this disease, it need to establish a stable and repeatable canine sepsis model that is in line with the clinical characteristics of the disease.The study selected 12 local dogs and randomly divided into three groups: rapid bolus injection group(Group A), continuous infusion group within 30 min(Group B) and continuous infusion group(Group C).Then, the lipopolysaccharides(LPS) with 2 mg · kg~(-1) were injected through the brachial vein in different modes of administration, thus the model was fully established.During the modeling period, body temperature(T), respiratory rate(RR), heart rate(HR) and mean arterial pressure(MAP) were monitored at 0, 10, 20, 30, 40, 50 min and 1, 2, 3, 4, 5, 6, 7, 8, 9, 12 and 24 h.Blood was collected from the canine brachial vein at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 12 and 24 h, respectively, for the detection of white blood cells(WBC).The test showed that the values of T, RR, HR, MAP, WBC of the dogs in group A all changed but did not exceed the normal range, and the clinical symptoms were not significant.There were no significant changes in the values of RR, HR, T, MAP and WBC of the dogs in Group B, and the clinical symptoms were not significant.The value of T of the dogs in Group C were significantly increased at 40 min(p0.05), which reached the fever standard and lasted for 7 h; the value of RR increased significantly at 20 min(p0.05), and a downward trend could be observed at 12 h, then it returned to normal at 24 h; the value of HR increased significantly at 50 min(p0.05) and recovered at 8 h; the value of HR decreased significantly at 20 min(p0.05), which remained at 12 h(p0.05), and returned back to normal at 24 h; the value of WBC decreased significantly from 1 h to4 h(p0.05), which was lower than the normal value, and increased significantly at 24 h(p0.01); all of the four dogs in this group had clinical symptoms such as vomiting, diarrhea and depression.Based on the above results, the changes of indexes and clinical symptoms in Groups A and B did not meet the standards of sepsis.After a long-term continuous intravenous infusion of LPS, the experimental dogs in Group C showed varying degrees of clinical symptoms, such as vomiting, diarrhea and depression one after another.The indexes and clinical symptoms reached the sepsis standard about 3 h after infusion.In brief, this model not only had good stability and good regularity of repeatability, but also lasted for a long time and could be suitable for other subsequent studies.     

Effects of Yimu Shenghuasan Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows

In order to investigate the mode of action ofYimu Shenghuasan preparation in endometrial cells of dairy cows, the primary cultured endometrial cells in cows were isolated and the inflammatory models were made by lipopolysaccharide （LPS） induction. The inflammatory cells were treated with gradient concentration of herbal medicine preparation, Yimu Shenghuasan for 48 and 72 h. The expression of cytochrome P450 （CYP450） was detected by Western blot. The amounts of IgG and lgA in sera were also detected in the endometritis of dairy cows. The expression level of CYP450 in the endometrial cells of dairy cow was increased gradually, and the amounts of IgG, IgA were increased significantly as compared with those in the control group. The expression level of CYP450 in the inflammatory cells was increased significantly in the treatment of 2 000 μg mL^-1 of Yimu Shenghuasan after 48 h of treatment.     

Study on Isolation, Passage, Cryopreservation and Histology of Mouse Fibroblasts

The embryonic ages were determined for the best preparation of mouse fibroblasts. Four methods were adapted to verify cryopreservation of mouse fibroblasts. The results showed that embryonic cryopreserving method was best one with 0.86 of thawing viability. The embryos from 13-14d pregnant mouse were superior to 11-12d and 15-16d in isolating, growing, laying and living. The first 6 generations were better than following ones in the same aspects above. Cell laying time became longer and vailable time became shorter after the sixth generation. With culture time increasing, fibroblast nuclear size became larger, fibrous filament appeared among fibroblasts, and macrocyst vesicle with floccule appeared in the cells. Cyst vesicle structure with pyknotic granule appeared in 24h cultured fibroblasts and macrocyst vesicle also appeared in passaging fibroblasts.     

The Special Expression and Comparison of the c-kit Protein in Spermatogenesis of Three Species of Locusts of Arcypteridae

The aim of this study was to elucidate the expression and regulation of the c.kit protein in spermatogenesis of locusts. Immunohistochemistry and biological statistics were used to investigate the expression of the c-kit protein in four representative phases of spermatogenesis of three dominant species of locusts of Arcypteridae （Orthoptera： Acridoidea）, namely, Omocestus viridulus （Linnaeus）, Euchorthippus unicolor （Ikonn.）, and Euchorthippus vittatus Zheng, and so on, in Siping area of Jilin Province, China. The results revealed the following： （1） There was weak positive expression of the c-kit protein in spermatogonia and the positive granules were thinner; （2） there was a strong positive expression of the c-kit protein in primary spermatocyte and the positive granules became the largest than in all developmental stages; （3） the c-kit protein positive expression became stronger in secondary spermatocyte, while the positive granules became thinner; （4） there was strong positive expression of the c-kit protein and the positive granules were thinner in mature sperm, which were distributed on its head and tail; （5） there were strong positive protein granules massing at the end of spermary; （6） the positive intensity of the c-kit protein in spermatogenesis was significantly different among different species of locusts. The data suggested that the c-kit protein may play a crucial role in spermatogenesis as well as maintain the physiological action of sperms and fertilization, regulate the developmental speed of spermatogenesis, and/or maintain species isolation, etc.     

Preparation,cryopreservation and in vitro culture of spermatogonial stem cells of new born calves

The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem cells. As for enzymolysis of testis tissue, two digestive method A and B. The cryopreservation cooling procedure was that spermatogonial stem cells was equilibrated at 4℃ for lh, at -20℃ for lh, at -40℃ for 2h, at -80℃ overnight and then stored in liquid nitrogen. The different concentration of three eryoprotectants were compared. The results indicated that both method A and method B achieved the same high motility rates of cells with method B needing time longer, calf spermatogonial stem cells could be well cryopreserved by the stage cooling procedure. Satisfactory cryopreservative results were gotten by adding 10% DMSO or 10% EG to cryopreservative medium, the former was better and adding sucrose could improved cryopreservative effect. The culture behaviors of spermatogonial stem cells kept the same before and after cryopreservation. It was concluded that an effective method was verified for preparation, cryopreservation and culture of new calf spermatogonial stem cells.     

The Effect of Yucca schidigera Extract on Ruminal Fermentation and Parameters Traits in Sheep

In a completely randomized block design experiment, 16 ruminally cannulated sheep (40 ± 2.1 kg)fed a 50% concentrate 50% forage diet (DM basis) were given intraruminal doses of powdered Yucca schidigera extract (YSE). Doses of 0 (control), 100, 200, or 300 mg kg-1 diet were given at 8 p.m. and 4 a.m. On 15, 16, and 17 d of the experimental period, ruminal contents were sampled 0, 2, 4, 6, and 8 h after dosing, and blood samples were collected at the end of the experiment (18th and 19th d). Acidity was not affected (P＞0.05) by the addition of YSE. Compared with the control, ruminal propionate concentration was increased by 29.4 and 29.8% (P＞0.05) and the acetic acid concentration was decreased by 15.1 and 19.8% (P＞0.05) at 4 and 6 h after YSE (300 mg kg-1) dosing, respectively. Ruminal ammonia concentration in the first 2 h after feeding was higher (P＜0.05) in the sheep that did not receive YSE (increased by 17.57 mg 100 mL-1) than in those that received 200 mg kg-1 (increased by 6.77 mg 100 mL-1) or 300 mg kg-1 (increased by 6.50 mg 100 mL-1) YSE. Protozoan populations in the rumen were lower (P ＜ 0.05) in the animals that received 300 mg kg-1 of YSE compared with the control. All serum parameters of the four groups were in the normal range and were similar among the treatment groups (P＞0.05), after being fed for 19 d with different doses of YSE. The effect of YSE on ruminal ammonia concentration likely resulted from a decreased concentration of protozoan populations and, presumably, from ammonia binding by YSE.     

Effects of Sub-chronic Aluminum Exposure on Renal Pathologic Structure in Rats

A total of 40 Wistar rats, weighing 130-140 g, were allocated randomly into four groups. They were orally administrated with 0 (control group, GC), 64.18 (low-dose group, GL), 128.36 (middle-dose group, GM), and 256.72 (high-dose group, GH) mg aluminum chloride (AlCl3) per kilogram body weight in drinking water for 120 days. Kidney coefficient and aluminum (Al) concentrations in blood and kidney were determined, and renal autopsy and histological changes were observed. The results showed that kidney coefficient in all Al-treated groups were obviously lower than that in GC (P<0.01) and there was a dose-effect relationship. The kidneys were solid, lusterless and pale brown with white necrosis point on surface. Under electron microscope, renal cortex became thin, the renal tubule was narrowed and the epithelium dissolved; the renal glomerulus became atrophied and the glomerular became vasodilator. The Al concentrations in blood and kidney were higher in all Al-treated rats than those in GC (P<0.01), and there was a dose-effect relationship. The results indicated that sub-chronic Al exposure could lead to Al accumulation in kidney, restrain the development of kidney and cause the pathologic damage in rats.     

Expression and Purification of Goose HSP70 and Compound Formation with Virus Polypeptide

The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty healthy 6-week-old male Wulong geese were selected and randomly divided into three groups. The control group was slaughtered without heat treatment. Treatment group 1 was shocked with an acute heat treatment at （42 ± 1）℃ for 5 h before they were immediately slaughtered. Treatment group 2 was kept for 12 h after the heat treatment under normal conditions in order to recover and was then slaughtered. Cardiac tissue was taken in order to make paraffin sections for the immunohistochemistry experiment and the liver tissue was used to purify HSP70. The geese heart HSP70 expression differences in the three groups were determined and at the same time the experiments of HSP70 purification and appraisal in the liver tissue were carried on. HSP70 purification and synthesis of HBV PreS1 multi-peptides unified the complex, which was determined by bi-specific antibody enzyme-linked immune sandwich assay. The results indicated that widespread HSP70 positive pellets in the cardiac muscle were found under hot shock conditions. HSP70 expression in the treatment group 1 was centered in the karyotheca and its periphery, but in treatment group 2, it was centered in the surrounding cell membrane. The HSP70 purification could be obtained through two sets of purification plans; both the synthesis peptide and the HSP70 purification form the complex under certain conditions. The double antibody sandwich ELISA technique was applied to detect if the complex had been formed. Positive results showed that the complex was formed. The specific expression of HSP70 under heat shock shifted with time, suggesting that HSP70 possibly had some function in cell protection. High-purity HSP70 protein can be obtained under low-pressure chromatography conditions, and in comparison with each other, it was better in the flow of the molecular     

Electronic Scanning of Uterine Endometrium in Postpartun Cow

Two postpartum cows were used to study the ultrastructural changes of uterine endometrium by using scanning electron microscope,The results showed that the process of repair of uterine endometrium after calv-ing was demonstrated by scanning electron microscope through a series of endometrium biopsy.Some part of the endometrium was damaged after calving and its adjacent endometrium cells became necrosis and exfoliated during the first 7days post-partum;the cilium and microvillus of the epithelial cell in the umdamaged area of the endomketrium disappeared,By26days postpartum the damaged area reduced and the cilium and microvillus increased in their numbers.The damaged tissues were all repaired by day60postpartum.