Chemosensitivity to doxorubicin in primary cells derived from tumor of FVB/N-Trp53tm1Hw1 with TALEN-mediated Trp53 mutant gene.

BACKGROUND: To evaluate the chemosensitivity to doxorubicin (DOX) in two primary cells derived from a tumor of FVB/N-Trp53tm1Hw1 knockout (KO) mice with TALEN-mediated Trp53 mutant gene, we evaluated the cell survivability, cell cycle distribution, apoptotic cell numbers and apoptotic protein expression in solid tumor cells and ascetic tumor cells treated with DOX. RESULTS: The primary tumor cells showed a significant (P < 0.05) defect for UV-induced upregulation of the Trp53 protein, and consisted of different ratios of leukocytes, fibroblasts, epithelial cells and mesenchymal cells. The IC50 level to DOX was lower in both primary cells (IC50 = 0.12 µM and 0.20 µM) as compared to the CT26 cells (IC50 = 0.32 µM), although the solid tumor was more sensitive. Also, the number of cells arrested at the G0/G1 stage was significantly decreased (24.7-23.1% in primary tumor cells treated with DOX, P < 0.05) while arrest at the G2 stage was enhanced to 296.8-254.3% in DOX-treated primary tumor cells compared with DOX-treated CT26 cells. Furthermore, apoptotic cells of early and late stage were greatly increased in the two primary cell-lines treated with DOX when compared to same conditions for CT26 cells. However, the Bax/Bcl-2 expression level was maintained constant in the primary tumor and CT26 cells. CONCLUSIONS: To the best of our knowledge, these results are the first to successfully detect an alteration in chemosensitivity to DOX in solid tumor cells and ascetic tumor cells derived from tumor of FVB/N-Trp53tm1Hw1 mice TALEN-mediated Trp53 mutant gene.

T-cell lymphoma infiltrating the uterus and ovaries of a Golden Retriever: a case report.

BACKGROUND: To the best of our knowledge, this is the first report of female genital system infiltration of T-cell lymphoma in veterinary literature. CASE PRESENTATION: A 1.5-year-old, intact female Golden Retriever was referred due to melena and hyporexia that lasted for three weeks. Fever (40.5℃), tachycardia, tachypnoea, pale mucous membranes, and purulent vaginal discharge were identified on physical examination. Blood analyses revealed leucocytosis, anaemia, hypoalbuminemia, and increased lactate and C-reactive protein levels. On abdominal radiography, the small intestine was moderately deviated because of an oval-shaped mass (13 cm × 8.7 cm) located in the mid-abdomen. An enlarged tubular-shaped structure that had the opacity of soft tissue located in dorsal to the bladder to the middle of the abdomen, and an oval-shaped mass (5.28 cm × 3.26 cm), which was suspected to be a medial iliac lymph node located at the sixth to seventh lumbar level. Abdominal ultrasonography revealed gas and fluid in the lumen of the uterine horn with a severely thickened wall, round enlarged lymph nodes around the genitourinary system, and free fluid in the abdominal cavity. Based on these results, pyometra was suspected, and an exploratory laparotomy was performed for ovariohysterectomy. The resected ovary and uterus were macroscopically hypertrophied. Histopathological examination of the ovary and uterus revealed neoplastic proliferation of large round cells with strong immunoreactivity for CD3, indicating T-cell lymphoma. Therefore, the young dog was diagnosed with genital lymphoma. CONCLUSIONS: The present report describes T-cell lymphoma infiltrating the uterus and ovaries in a young dog, which is rarely diagnosed and could aid in the differential diagnosis of genital diseases in young dogs.

Factors affecting reproductive outcomes in lactating dairy cows that undergo presynchronization-Ovsynch and successive resynchronization programs.

This study was designed to identify the factors affecting reproductive outcomes in lactating dairy cows undergoing Presynchronization-Ovsynch and successive resynchronization programs. Data were collected from 1,458 cows on 11 dairy farms, regarding nutrition, health, reproduction, and specifically reproductive programs used for the first timed artificial insemination (TAI; Modified Presynch-Ovsynch or Modified Double-Ovsynch) and for re-inseminations (Ovsynch or PreGnRH-Ovsynch); as well as the weather. Body condition score (BCS) and timing of TAI affected (P < 0.01) the probability of pregnancy per AI after first TAI. Cows with a BCS ≥3.25 were more likely (P < 0.01) to be pregnant at 31 (odds ratio [OR]: 1.85) and 49 (OR: 1.93) days after the first TAI than cows with BCS ≤2.75. Cows inseminated during months of heat stress (June to September) were less likely (P < 0.01) to be pregnant at 31 (OR: 0.74) and 49 (OR: 0.68) days after first TAI than those inseminated during months of no heat stress (October to May). Cows that had a pre- or postpartum disorder were more likely to lose their pregnancy (OR: 2.17, P < 0.01) than those that did not. Cows inseminated ≥76 days after calving (OR: 1.67, P < 0.1) or during the months of heat stress (OR: 2.02, P < 0.05) were also more likely to lose pregnancy than cows inseminated ≤75 days after calving or during the months of no heat stress. Following successive resynchronizations, cows with a third (OR: 1.32, P < 0.1) or fourth (OR: 1.68, P < 0.05) TAI were more likely to be pregnant than those with a second TAI. Cows with BCS ≥3.25 at the beginning of resynchronization were more likely (OR: 1.49, P < 0.05) to be pregnant than cows with BCS ≤2.75. Finally, the likelihood of being pregnant by 210 days postpartum was positively associated with a favorable BCS, but negatively associated with the presence of a pre- or postpartum disorder and a larger number of days to first TAI following calving (≥76 vs. ≤75 days). In conclusion, high BCS has beneficial effects during the entire reproductive period, whereas TAI during heat stress, the presence of a pre- or postpartum disorder, a larger number of days to first TAI following calving, and TAI number (second or ≥ fifth) adversely affects the outcomes associated with pregnancy during the early or later breeding periods in lactating dairy cows undergoing Presynchronization-Ovsynch and successive resynchronization programs.

Selective use of a modified pre-synchronization-Ovsynch and resynchronization reproductive strategy in dairy herds: A field application study.

We aimed to evaluate whether a modified pre-synchronization-Ovsynch (MPO) and resynchronization method, adjusted for the timing of biweekly reproductive consulting visits, could be implemented successfully under field conditions in dairy herds. Forty-five days after calving (calving: Day 0), 1,182 cows were assigned randomly to Ovsynch, MPO or control groups. (1) Cows were administered GnRH on Day 65, PGF2α on Day 72 and GnRH 56 hr later, followed by timed artificial insemination (TAI) 16 hr later (Ovsynch, n = 380). (2) Cows with a CL were administered PGF2α (n = 204) and cows without were administered GnRH (n = 199) on Day 45. Then, each cow was administered PGF2α on Day 55 (9 to 12 days later), GnRH on Day 58 and underwent Ovsynch 7 days later (selective MPO [sMPO] n = 403). (3) The remaining cows underwent AI when oestrus was detected (EDAI, n = 399). The probabilities of pregnancy per AI 30 and 45 days after the first AI were higher in the sMPO group (odds ratios 1.99 and 1.92, p < .0001) than in the EDAI group. In addition, the probability of pregnancy by 105 days postpartum tended to be higher (p < .1) in the sMPO group than in the EDAI and Ovsynch groups, which shortened the mean interval from calving to pregnancy in the sMPO group (3.4 and 4.2 days, respectively) than in the EDAI and Ovsynch groups. In the sMPO group, the interval between the first and second treatments during pre-synchronization did not affect the probability of pregnancy per AI after the first AI. The probability of pregnancy per AI after the second AI did not differ between the Ovsynch, sMPO and EDAI groups. In conclusion, the sMPO programme, adjusted for the timing of biweekly reproductive consulting visits, followed by resynchronization, could be successfully adopted in field conditions, and is associated with good reproductive performance in dairy herds.

Comparison of cisplatin-induced anti-tumor response in CT26 syngeneic tumors of three BALB/c substrains.

BACKGROUND: To determine whether the background of BALB/c substrains affects the response to anti-tumor drugs, we measured for alterations in tumor growth, histopathological structure of the tumor, and expressions of tumor-related proteins in three BALB/c substrains derived from different sources (BALB/cKorl, BALB/cA and BALB/cB), after exposure to varying concentrations of cisplatin (0.1, 1 and 5 mg/kg). RESULTS: Cisplatin treatment induced similar responses for body and organ weights, serum analyzing factors, and blood analyzing factors in all BALB/c substrains with CT26 syngeneic tumor. Few differences were detected in the volume and histopathological structure of the CT26 tumor. Growth inhibition of CT26 tumors after exposure to cisplatin was greater in the BALB/cB substrain than BALB/cKorl and BALB/cA substrains, and a similar pattern was observed in the histopathological structure of tumors. However, the expression levels of other tumor-related factors, including Ki67, p27, p53, Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), caspase-3 (Cas-3), matrix metallopeptidase 2 (MMP2) and vascular endothelial growth factor (VEGF) proteins, were constantly maintained in the tumors of all three substrains after cisplatin treatment. A similar decrease pattern was observed for the expressions of inflammatory cytokines, including interleukin (IL)-1ß, IL-6 and IL-10, in the CT26 tumors of the three BALB/c substrains. CONCLUSIONS: Taken together, results of the present study indicate that the genetic background of the three BALB/c substrains has no major effect on the therapeutic responsiveness of cisplatin, except growth and histopathology of the CT26 syngeneic tumor.

Lateral Flank Approach for Ovariohysterectomy in a Lion (Panthera leo) with a Ruptured Pyometra.

An 8.5-year-old intact female lion (Panthera leo) with a history of vomiting, lethargy, and anorexia was referred to our institution. On physical examination, the lion weighed 180 kg and had a rectal temperature of 40 °C. Blood analysis showed mild neutrophilic leukocytosis, and abdominal ultrasonography revealed an enlarged uterus with echogenic fluid. Based on the clinical signs and hematologic and ultrasonographic findings, the lion was tentatively diagnosed with pyometra. Ovariohysterectomy was performed using the lateral flank approach to avoid complications that can occur with ventral celiotomy. Surgery was performed successfully despite unexpected rupture of the uterus which had occurred before the surgery. The lion recovered uneventfully and continued to do well at the 1 year follow up after surgery. To the best of our knowledge, this is the first reported case of ovariohysterectomy with the lateral flank approach in a lion with pyometra. Our report suggests that ovariohysterectomy with the lateral flank approach could be considered as an alternative method for pyometra in wild big cats with risk of complications at the surgical site.

Reproductive performance following a modified Presynch-Ovsynch, Double-Ovsynch, or conventional reproductive management program in Korean dairy herds.

We aimed to compare the reproductive performance of dairy cattle following a modified Presynch-Ovsynch, Double-Ovsynch, or conventional reproductive management program (CRMP). On Day 30 (±5) after calving (calving = Day 0), 960 lactating cows were assigned randomly to one of two presynchronization treatments, before the synchronization of ovulation (Ovsynch) or CRMP without presynchronization. Cows were administered prostaglandin F2α (PGF2α) on Days 38 (±5) and 52, and gonadotropin-releasing hormone (GnRH) on Day 55 (Presynch-G-Ovsynch group, n = 333); or GnRH on Day 45 (±5), PGF2α on Day 52, and GnRH again on Day 55 (Double-Ovsynch group, n = 307). Thereafter, each cow underwent Ovsynch 7 days later: GnRH on Day 62, PGF2α on Day 69, and GnRH again 56 h later, followed by timed artificial insemination (AI) 16 h later. The remaining cows underwent AI when estrus was detected or Ovsynch (CRMP group, n = 320). In a subset of cows (each n = 40) in the two presynchronization groups, blood collections and ovarian ultrasonography were performed on Days 30 (±4), 52, 62, and 69, and uterine cytology on Days 30 (±4) and 52. The probabilities of pregnancy per AI 32 and 60 days after the first AI were higher in the Presynch-G-Ovsynch (odds ratio [OR]: 1.89 and 1.81, P < 0.01) and Double-Ovsynch (OR: 1.63 and 1.60, P < 0.05) groups than in the CRMP group. The likelihood of pregnancy by 210 days postpartum was higher (P < 0.05) in Presynch-G-Ovsynch (hazard ratio [HR]: 1.29) and Double-Ovsynch (HR: 1.31) groups than in the CRMP group, whereas the least square mean number of inseminations per conception was lower (P < 0.05) in Presynch-G-Ovsynch (1.8) and Double-Ovsynch (1.8) groups than in the CRMP group (2.1). The percentages of cows with serum progesterone concentrations ≥1.0 ng/mL or with a corpus luteum (CL) did not differ between the groups (P > 0.1) on Days 30, 62, and 69, but were lower (P < 0.05) on Day 52 in the Presynch-G-Ovsynch than in the Double-Ovsynch groups. However, the diameter of the dominant follicles, the proportion of neutrophils in uterine cytological samples, and the prevalence of an accessory CL on Day 69 did not differ (P > 0.1) between the two groups. In conclusion, both the Presynch-G-Ovsynch and Double-Ovsynch programs improved reproductive performance vs. CRMP in smallholder Korean dairy herds, and there were no differences in the ovarian endocrine and structural dynamics, or uterine health, between the two programs involving presynchronization.

Effect of Minoxidil on Trabecular Outflow via the Paracellular Pathway.

PURPOSE: To investigate the pathway and effects of minoxidil on trabecular outflow in cultured human trabecular meshwork (TM) cells. METHODS: After exposing primarily cultured TM cells to 0, 10, 50, or 100 µM minoxidil sulfate (MS), trabecular outflow was assessed by measuring TM cell monolayer permeability to carboxyfluorescein and transepithelial electrical resistance. To assess the pathway of permeability changes, caveolin-1, occludin, and claudin-5 levels were measured via western blot. Generation of reactive oxygen species (ROS) was measured using the dichlorofluorescein diacetate assay. To assess the involvement of nitric oxide (NO) in minoxidil-induced permeability increase, the degrees of endothelial nitric oxide synthase mRNA expression and NO production were measured with reverse transcription polymerase chain reaction and Griess assays, respectively. Permeability was also measured with co-exposure to 50 µM N-acetyl cysteine. RESULTS: MS significantly increased TM cell monolayer permeability (p < 0.05) and decreased transepithelial electrical resistance (p < 0.05). MS decreased the degree of endothelial nitric oxide synthase mRNA expression but did not affect NO production. MS decreased occludin and claudin-5 levels but did not affect caveolin-1 level. MS at 100 µM increased the generation of ROS, and MS-induced permeability increase was attenuated after co-exposure to 50 µM N-acetyl cysteine. CONCLUSIONS: Minoxidil may preferentially increase trabecular permeability via a paracellular pathway by downregulation of tight junction proteins. This minoxidil-induced permeability through the TM may be mediated by generation of ROS.

Heat Shock Factor 1 Predicts Poor Prognosis of Gastric Cancer.

PURPOSE: Heat shock factor 1 (HSF1) is a key regulator of the heat shock response and plays an important role in various cancers. However, the role of HSF1 in gastric cancer is still unknown. The present study evaluated the function of HSF1 and related mechanisms in gastric cancer. MATERIALS AND METHODS: The expression levels of HSF1 in normal and gastric cancer tissues were compared using cDNA microarray data from the NCBI Gene Expression Omnibus (GEO) dataset. The proliferation of gastric cancer cells was analyzed using the WST assay. Transwell migration and invasion assays were used to evaluate the migration and invasion abilities of gastric cancer cells. Protein levels of HSF1 were analyzed using immunohistochemical staining of tissue microarrays from patients with gastric cancer. RESULTS: HSF1 expression was significantly higher in gastric cancer tissue than in normal tissue. Knockdown of HSF1 reduced the proliferation, migration, and invasion of gastric cancer cells, while HSF1 overexpression promoted proliferation, migration, and invasion of gastric cancer cells. Furthermore, HSF1 promoted the proliferation of gastric cancer cells in vivo. In Kaplan-Meier analysis, high levels of HSF1 were associated with poor prognosis for patients with gastric cancer (p=0.028). CONCLUSION: HSF1 may be closely associated with the proliferation and motility of gastric cancer cells and poor prognosis of patients with gastric cancer. Accordingly, HSF1 could serve as a prognostic marker for gastric cancer.

Berberine Suppresses Fibronectin Expression through Inhibition of c-Jun Phosphorylation in Breast Cancer Cells.

PURPOSE: The exact mechanism regulating fibronectin (FN) expression in breast cancer cells has not been fully elucidated. In this study, we investigated the pharmacological mechanism of berberine (BBR) with respect to FN expression in triple-negative breast cancer (TNBC) cells. METHODS: The clinical significance of FN mRNA expression was analyzed using the Kaplan-Meier plotter database (http://kmplot.com/breast). FN mRNA and protein expression levels were analyzed by real-time polymerase chain reaction and western blotting, respectively. RESULTS: Using publicly available clinical data, we observed that high FN expression was associated with poor prognosis in patients with breast cancer. FN mRNA and protein expression was increased in TNBC cells compared with non-TNBC cells. As expected, recombinant human FN significantly induced cell spreading and adhesion in MDA-MB231 TNBC cells. We also investigated the regulatory mechanism underlying FN expression. Basal levels of FN mRNA and protein expression were downregulated by a specific activator protein-1 (AP-1) inhibitor, SR11302. Interestingly, FN expression in TNBC cells was dose-dependently decreased by BBR treatment. The level of c-Jun phosphorylation was also decreased by BBR treatment. CONCLUSION: Our findings demonstrate that FN expression is regulated via an AP-1-dependent mechanism, and that BBR suppresses FN expression in TNBC cells through inhibition of AP-1 activity.

Detailed Anatomy of Osteoperiosteal Ligamentous Structures in the Forehead.

This study aimed to present the anatomic characteristics of osteoperiosteal ligamentous attachments of the forehead and provide a better understanding of lateral eyebrow descent for safe and effective foreheadplasty. Anatomic dissections of the face were performed in 10 fresh Korean adult cadavers (20 hemifaces) using 2.5× magnification surgical loupes. Supraorbital, inferomedial orbital, and frontonasal osteoperiosteal ligamentous attachments were identified as fibrous tissues originating from a bone, and their tensile strengths were measured. The supraorbital osteoperiosteal ligamentous attachment had medial and lateral parts. It can be classified into 4 subtypes. It was located 11.0 ±â€Š6.6 mm lateral to the midline and 9.2 ±â€Š12.3 mm superior to the superior orbital margin. The inferomedial orbital osteoperiosteal ligamentous attachment was located 16.2 ±â€Š3.9 mm lateral to the midline and 2.2 ±â€Š2.7 mm inferior to the superior orbital margin, whereas the frontonasal osteoperiosteal ligamentous attachment was located 5.4 ±â€Š2.3 mm lateral to the midline and 1.4 ±â€Š8.5 mm superior to the superior orbital margin. Tensile strengths of all the osteoperiosteal ligamentous attachments in the forehead were above 10 N. These results indicate that osteoperiosteal ligamentous attachments develop in the rather medial region of the eyebrow and have a tensile strength adequate enough to maintain the medial eyebrow. Thus, the current study provides surgeons with detailed anatomic information that can be used as a valuable reference for forehead rejuvenation procedures.

Facial Nerve Supply to the Orbicularis Oculi around the Lower Eyelid: Anatomy and Its Clinical Implications.

BACKGROUND: The motor innervation of the lower orbicularis oculi has not been clearly established. There is a discrepancy between anatomical descriptions and clinical outcomes of the motor innervation of the pretarsal orbicularis oculi muscle. Therefore, the purposes of this study were to identify every motor and sensory nerve of the lower eyelid, and to reveal the detailed motor nerve pathways toward the medial canthal area. METHODS: Fresh cadaver dissections were performed on 50 hemifaces under a surgical microscope. Submuscular and intramuscular nerves of the lower eyelid were identified, and the pathways of facial nerves that ran toward the medial canthus were traced. RESULTS: Vertical submuscular nerves at the lower eyelid originated from the infraorbital foramen, indicating that all were sensory nerves. The zygomatic branch of the facial nerve traveled obliquely through the anterior cheek and supplied the orbicularis oculi of the lower eyelid and the medial portion of the upper eyelid. Its route was defined as a clinically useful line, the medial orbicularis motor line. In addition, the nerve innervating the pretarsal orbicularis oculi arose at the superomedial preseptal area and extended horizontally and laterally. Interestingly, the angular nerve appeared not to innervate the palpebral orbicularis oculi. CONCLUSIONS: In the lower eyelid, the vertical sensory and the oblique motor nerve supplies are independent and clearly distinguished in aspect of their own routes. The medial orbicularis motor line represents the motor route to the medial portion of the orbicularis oculi. These results might provide valuable knowledge about surgical anatomy for safe lower blepharoplasty with or without midface lift.

Three Skin Zones in the Asian Upper Eyelid Pertaining to the Asian Blepharoplasty.

BACKGROUND: Natural looking double fold is an essential and aesthetically pleasing masterpiece in Asian blepharoplasty. This study aims to emphasize the 3 skin zone concept in the Asian upper blepharoplasty. METHODS: The authors examined the anterior lamella of each skin zone microscopically by performing 31 double-eyelid surgeries and 11 infrabrow lifts. Characteristics of dermal components, subcutaneous tissue, and outer fascia of OOM (OFOOM) at each skin zone were documented. The authors evaluated the vertical scales of each skin zone in young and aged Asian patients who visited the first author's clinic for the primary or secondary upper blepharoplasty with ×3.5 magnifying surgical loupe. RESULTS: The thickness of OOM had no difference among zones 1, 2, and 3. The skin and subdermal tissue had varying characteristics according to its skin zone. At zone 1, it seemed that only thin skin was on the OOM. The anterior lamella of zone 2 seemed to consist of skin, white fascia (OFOOM) including a venous network, and OOM in a gross field. At zone 3, thick skin, thick subcutaneous fatty layer, and OOM were magnified. The OFOOM of zone 3 was not significantly identified due to a sticky adherence with OOM. At the point of vertical scales of skin zone, good eyelids have lower zone 3 ratio and higher zones 1 and 2 ratio with qualified topographic condition. CONCLUSION: The authors classified the Asian upper eyelid as with 3 skin zones. Based on its anatomical investigation, the authors can afford anthropometric data and supplemental theory for the creation of aesthetic folds.

Bilayered Structure of the Superficial Facial Fascia.

Background: The superficial facial fascia comprises the superficial musculoaponeurotic system (SMAS) and the temporoparietal fascia (TPF) and is regarded as a continuous monolayer. However, some evidence indicates that the superficial facial fascia consists of 2 layers in specific areas. Objectives: The authors evaluated the superficial facial fascia for bilayered regions. Methods: Twenty fresh cadavers (40 hemifaces) were dissected to observe the superficial facial fascia. Twelve cadavers were dissected to assess tensile strengths of the superficial and deep layers of the SMAS. Specimens were obtained from 2 cadavers for histologic analysis. Results: The SMAS and TPF were separable into superficial and deep layers, with intervening areolar tissue. The deep TPF was continuous with the deep SMAS inferiorly and the subgalea anteriorly. The superficial orbicularis oculi was invested by the superficial SMAS, whereas the deep orbicularis and the platysma were invested by the deep SMAS. Thus, 2 key structures addressed in facial rejuvenation are positioned in different surgical planes. Conclusions: Study results support the belief that the superficial facial fascia comprises 2 layers, with the superficial orbicularis oculi and platysma invested by different layers. These findings have implications for facial rejuvenation techniques that involve management of the SMAS and TPF.

Detailed anatomy of the retaining ligaments of the mandible for facial rejuvenation.

PURPOSE: To present anatomical characteristics of three true ligaments of the mandible and better understanding of jowl formation. MATERIALS AND METHODS: Facial dissections were performed in 10 fresh cadavers (20 hemifaces) using 2.5 magnifying surgical loupes. Mental ligament, medial mandibular ligament, and mandibular osteocutaneous ligament were identified as thick fibrous tissue originating from the mandible. RESULTS: The location of the mental ligament was 9.1 ± 2.8 mm lateral to the midline and 8.4 ± 3.8 mm superior to the inferior border of the mandible. The medial mandibular ligament was located 28.5 ± 4.9 mm lateral to the midline and 8.4 ± 2.7 mm superior to the inferior border of the mandible. The mandibular osteocutaneous ligament was located 51.1 ± 4.5 mm lateral to the midline and 9.1 ± 3.1 mm superior to the inferior border of the mandible. CONCLUSIONS: We investigated location and tension of two unfamiliar ligaments in the mandible, the mental and medial mandibular ligaments medial to the mandibular osteocutaneous ligament, and confirmed them as true ligaments by histology. Our findings suggest that the cause of the jowl formation is mainly descent of the cheek fat compartments, but not laxity of mandibular osteocutaneous ligament.

Effects of gonadotropin-releasing hormone administration or a controlled internal drug-releasing insert after timed artificial insemination on pregnancy rates of dairy cows.

This study investigated the effects of gonadotrophin-releasing hormone (GnRH) administration (Experiment 1) and a controlled internal drug-releasing (CIDR) insert (Experiment 2) after timed artificial insemination (TAI) on the pregnancy rates of dairy cows. In Experiment 1, 569 dairy cows that underwent TAI (day 0) following short-term synchronization with prostaglandin F2α were randomly allocated into two groups: no further treatment (control, n = 307) or injection of 100 µg of gonadorelin on day 5 (GnRH, n = 262). In Experiment 2, 279 dairy cows that underwent TAI (day 0) following Ovsynch were randomly allocated into two groups: no further treatment (control, n = 140) or CIDR insert treatment from days 3.5 to 18 (CIDR, n = 139). The probability of pregnancy following TAI did not differ between the GnRH (34.4%) and control (31.6%, p > 0.05) groups. However, the probability of pregnancy following TAI was higher (odds ratio: 1.74, p < 0.05) in the CIDR group (51.1%) than in the control group (39.3%). Overall, CIDR insert treatment at days 3.5 to 18 increased pregnancy rates relative to non-treated controls, whereas a single GnRH administration on day 5 did not affect the pregnancy outcomes of dairy cows.

Effects of paracrine factors on CD24 expression and neural differentiation of male germline stem cells.

Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.

Relationships among ketosis, serum metabolites, body condition, and reproductive outcomes in dairy cows.

We determined the relationships among ketosis, serum metabolites, body condition, and reproductive disorders and performance in dairy cows. Blood samples from 213 dairy cows were collected at 4 and 2 weeks prepartum, just after calving, and at 1, 2, 4, 6, and 8 weeks postpartum to measure serum ß-hydroxybutyrate, nonesterified fatty acids (NEFAs), glucose, total cholesterol, urea nitrogen, aspartate aminotransferase, γ-glutamyltransferase, and progesterone concentrations. Cows were grouped on the basis of the ß-hydroxybutyrate concentration at 1 and/or 2 weeks postpartum into two groups: the ketotic group (≥1200 µmol/L, n = 59) and the nonketotic group (<1200 µmol/L, n = 154). The body condition score (BCS) was assessed simultaneously with blood collection. Clinical endometritis was diagnosed by observation of vaginal discharges (>50% pus), and subclinical endometritis was diagnosed by evaluation of uterine cytology (>18% neutrophils) at 4 weeks postpartum. Ovarian cysts were diagnosed by ultrasonography, and resumption of postpartum cyclicity was evaluated by progesterone concentrations (≥1 ng/mL) at 4, 6, and 8 weeks postpartum. In the ketotic group, NEFA levels were higher (P ≤ 0.0005), whereas glucose (P < 0.05-0.0005) and urea nitrogen levels (P < 0.05-0.01) were lower than those in the nonketotic group during the postpartum period. Aspartate aminotransferase levels were higher (P < 0.01) in the ketotic group than those in the nonketotic group at 2 weeks postpartum. The BCS of the ketotic group was higher than the nonketotic group during the prepartum (P < 0.001) and postpartum (P < 0.05-0.001) periods. The probabilities of clinical endometritis (odds ratio = 2.55) and ovarian cysts (odds ratio = 2.80) were higher (P < 0.05) in the ketotic group than those in the nonketotic group. The hazards of resumption of postpartum cyclicity by 8 weeks postpartum (hazard ratio = 0.67) and pregnancy by 360 days postpartum (hazard ratio = 0.68) were lower (P < 0.05) in the ketotic group. In conclusion, a higher BCS during prepartum and postpartum period and increased NEFA and aspartate aminotransferase levels, along with decreased glucose and urea nitrogen levels during postpartum, were associated with ketosis, increased reproductive disorders, and decreased reproductive performance in dairy cows.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.