Trends in oncology drug lags in Japan from 2001 to 2020: A cross-sectional study.

Anticancer drugs are essential in the treatment of serious diseases, but their applications are limited by drug lags. This study investigated the characteristics of anticancer drugs approved in Japan over the past 20 years and compared the drug lag trends between Japan and the US. We assessed the changes in drug lag between Japan and the US and the factors affecting the drug lags using publicly available data for anticancer drugs approved in Japan from January 2001 to December 2020. A total of 299 anticancer drugs were approved in Japan in the last 20 years. The approval lag median between the US and Japan was 498 days (16.6 months), peaking in 2002, and decreasing annually thereafter. The minimum approval lag was 173.5 days (5.7 months) in 2018. Multivariate regression analysis revealed that "global simultaneous strategy," "catch-up strategy," and "immunotherapy" are major factors shortening the drug lag. In the past decade, 226 anticancer drugs were approved in Japan. The drug lag for anticancer drugs between Japan and the US peaked in 2002, after which it declined sharply to less than a year. However, the lag was shortest in 2018.

Assessment of Surrogate End Point Trends in Clinical Trials to Approve Oncology Drugs From 2001 to 2020 in Japan.

Importance: A surrogate end point (SEP) is an end point used in clinical trials as an alternative for measuring the true clinical benefit. The use of SEPs in trials shortens their duration. Objectives: To investigate the use of SEPs in clinical trials to support the approval of anticancer drugs and to determine whether confirmatory studies that use overall survival (OS) as an end point are being conducted in Japan. Design, Setting, and Participants: In this cross-sectional study, drug approvals and background information were obtained from publicly available information, such as the Pharmaceuticals and Medical Devices Agency website, for anticancer drugs approved in Japan from January 2001 to December 2020. Data analysis was performed from September 2021 to March 2022. Main Outcomes and Measures: Characteristics of approved oncology drugs in Japan, end points for pivotal clinical trials, and outcomes of confirmatory trials using OS as an end point following drug approval. Results: There were 299 anticancer drugs approved in Japan during the study period. Of these, 142 (47.5%) were molecular-targeted drugs, the most common of which targeted non-small cell lung cancer. There were 111 (37.1%) anticancer drugs with orphan designation. From 2001 to 2005, OS was used as an end point in 1 approval (3.6%); however, from 2006 to 2020, OS was used in 86 approvals (31.7%). Of the 212 anticancer drugs approved on the basis of SEPs, confirmatory studies with OS as the end point were conducted for only 37 approvals (17.5%); for the remaining 175 approvals, studies are under way for 35 approvals (16.5%), were waivered for 75 approvals (35.4%), and were not conducted for 65 approvals (30.7%). Furthermore, in 20 drug approvals (9.4%), the conducted confirmatory studies were not effective in determining the OS, but the drugs were approved following re-examination. Conclusions and Relevance: The findings of this study suggest that starting from 2005, the use of OS as an end point has increased in studies supporting the approval of anticancer drugs in Japan. However, even after 2005, approximately two-thirds of these approvals were SEP based. Postmarketing surveillance studies of the true end points are necessary to validate the use of SEPs.

Invasive breast cancer with complete infarct necrosis: Presentation of case.

INTRODUCTION AND IMPORTANCE: Infarction occurs occasionally in benign mammary tumors but is extremely rare in breast cancer, with few cases having been reported. PRESENTATION OF CASE: A 53-year-old woman presented to our hospital with a mass and pain in the upper lateral region of the right breast. She underwent a needle biopsy and was histologically diagnosed as having invasive carcinoma. A ring-enhancing spherical mass was seen on contrast-enhanced computed tomography and magnetic resonance images. She underwent a right partial mastectomy with sentinel lymph node biopsy for T2N0M0 breast cancer. Macroscopically, the tumor was a yellow mass. Histopathologically, the site contained extensively necrotic tissue with foam cell aggregation, lymphocytic infiltration, and fibrosis in the periphery. No viable tumor cells were observed. The patient was followed up without postoperative chemotherapy or radiotherapy. CLINICAL DISCUSSION: Ultrasound examination performed before the biopsy showed blood flow inside the tumor, but a review of the histopathological tissue after surgery revealed generally low viability of the tumor cells in the biopsy specimen, and the possibility that the tumor had a strong tendency to be necrotic from the beginning was considered. It is presumed that some immunological mechanism was working. CONCLUSION: We have encountered a case of breast cancer with complete infarct necrosis. Infarct necrosis may be present if a contrast-enhanced image shows ring-like contrast.

Two cases of interparietal inguinal hernias undergoing laparoscopic treatment: a case series.

Interparietal inguinal hernia, an exceedingly rare type of inguinal hernia in which the hernia sac anatomically lies between the tissue layers of the abdominal wall, is difficult to diagnose from physical findings. Given the few reports on interparietal inguinal hernias, this condition has remained fairly unrecognized. Herein, we report the successful imaging and laparoscopic diagnoses as well as repair of an interparietal inguinal hernia. Atypical physical findings and computed tomography data help in the diagnosis of an interparietal inguinal hernia. The laparoscopic approach is useful and feasible for both the diagnosis and treatment of interparietal inguinal hernia.

Contralateral axillary lymph node metastasis in primary breast cancer: A case report.

INTRODUCTION: Contralateral axillary lymph node metastasis (CAM) is rare, especially in primary breast cancer. PRESENTATION OF CASE: A 71-year-old woman visited our hospital after noticing a mass in her right breast. A mass of 5 cm in diameter with skin infiltration was palpable on the medial side of the right breast. She underwent a needle biopsy and was diagnosed with invasive ductal carcinoma. On various imaging modalities, there were no distant metastases but bilateral axillary lymph node metastases were found. She underwent preoperative chemotherapy and showed a clinical partial response. After thorough discussion, she opted for surgery and underwent partial mastectomy of the right breast and bilateral axillary lymph node dissection. Histopathological examination revealed residual breast tumor and one metastatic axillary lymph node on each side. Postoperative radiotherapy and hormone therapy were performed. The patient is alive and recurrence-free as of 1 year after the start of treatment. CONCLUSION: CAM is often regarded as distant metastasis, but can be considered curable if there is no distant metastasis to other organs. CAM without distant metastasis to other organs should be treated with curative intent in order to avoid a disadvantage to the patient.

Unresectable esophageal cancer treated with multiple chemotherapies in combination with chemoradiotherapy: A case report.

BACKGROUND: Definitive chemoradiotherapy (dCRT) using cisplatin plus 5fluorouracil (CF) with radiation is considered the standard treatment for unresectable locally advanced T4 esophageal squamous cell carcinoma (ESCC). Recently, induction chemotherapy has received attention as an effective treatment strategy. CASE SUMMARY: We report a successful case of a 59-year-old female with unresectable locally advanced T4 ESCC treated by two additional courses of chemotherapy with CF after induction chemotherapy with docetaxel, cisplatin and fluorouracil (DCF) followed by dCRT. Initial esophagogastroduodenoscopy (EGD) detected a type 2 advanced lesion located on the middle part of the esophagus, with stenosis. Computed tomography detected the primary tumor with suspected invasion of the left bronchus and 90° of direct contact with the aorta, and upper mediastinal lymph node metastasis. Pathological findings from biopsy revealed squamous cell carcinoma. We initially performed induction chemotherapy using three courses of DCF, but the lesion was still evaluated unresectable after DCF chemotherapy. Therefore, we subsequently performed dCRT treatment (CF and radiation). After dCRT, prominent reduction of the primary tumor was recognized but a residual tumor with ulceration was detected by EGD. Since the patient had some surgical risk, we performed two additional courses of CF and achieved a clinically complete response. After 14 mo from last administration of CF chemotherapy, recurrence has not been detected by computed tomography and EGD, and biopsy from the scar formation has revealed no cancer cells. CONCLUSION: We report successful case with tumor remnants even after DCF and subsequent dCRT, for whom a complete response was finally achieved with two additional courses of CF chemotherapy. Additional CF chemotherapy could be one radical treatment option for residual ESCC after treatment with induction DCF followed by dCRT to avoid salvage surgery, especially for high-risk patients.

Pancreaticoduodenectomy after neoadjuvant chemotherapy for gastric cancer invading the pancreatic head: A case report.

BACKGROUND: Pancreaticoduodenectomy (PD) for advanced gastric cancer is rarely performed because of the high morbidity and mortality rates and low survival rate. However, neoadjuvant chemotherapy for advanced gastric cancer has improved, and chemotherapy combined with trastuzumab may have a preoperative tumor-reducing effect, especially for human epidermal growth factor receptor 2 (HER2)-positive cases. CASE SUMMARY: We report a case of successful radical resection with PD after neoadjuvant S-1 plus oxaliplatin (SOX) and trastuzumab in a patient (66-year-old male) with advanced gastric cancer invading the pancreatic head. Initial esophagogastroduodenoscopy detected a type 3 advanced lesion located on the lower part of the stomach obstructing the pyloric ring. Computed tomography detected lymph node metastasis and tumor invasion to the pancreatic head without distant metastasis. Pathological findings revealed adenocarcinoma and HER2 positivity (immunohistochemical score of 3 +). We performed staging laparoscopy and confirmed no liver metastasis, no dissemination, negative lavage cytological findings, and immobility of the distal side of the stomach due to invasion to the pancreas. Laparoscopic gastrojejunostomy was performed at that time. One course of SOX and three courses of SOX plus trastuzumab were administered. Preoperative computed tomography showed partial response; therefore, PD was performed after neoadjuvant chemotherapy, and pathological radical resection was achieved. CONCLUSION: We suggest that radical resection with PD after neoadjuvant chemotherapy plus trastuzumab is an option for locally advanced HER2-positive gastric cancer invading the pancreatic head in the absence of non-curative factors.

Bilateral nipple-sparing mastectomy and breast reconstruction in BRCA1 mutation-positive simultaneous bilateral breast cancer: A case study.

BACKGROUND: Mutation-positive patients who develop unilateral breast cancer require different treatments, such as prophylactic mastectomy of the contralateral breast, from those used for other breast cancer patients. If a mutation is found before surgery, it is necessary to consider a surgical procedure that includes reconstruction. For BRCA mutation-positive patients, a suitable treatment must be selected. In Japan, a test for BRCA mutation has been covered by health insurance since 2020, making it possible to preoperatively test patients who are suspected of being positive. We report a case of simultaneous bilateral breast cancer that was found to be BRCA mutation-positive preoperatively and underwent bilateral subcutaneous mastectomy and breast reconstruction. CASE PRESENTATION: A 57-year-old woman was admitted to our hospital after a breast cancer screening revealed a mass in the left breast. She had a family history of breast cancer, including her sister, aunt, and cousin. She was suspected of being malignant with a mass on both sides of her breast on imaging. She underwent needle biopsy and was diagnosed as having bilateral invasive ductal carcinoma, for which she was placed on preoperative chemotherapy. Due to the strong family history of bilateral breast cancer, the patient was recommended to undergo a BRCA gene-mutation test and she consented. The result was positive for BRCA1 mutation. Although it was judged that bilateral breast-conserving surgery was sufficiently possible, bilateral subcutaneous mastectomy and breast reconstruction were performed based on BRCA mutation-positive status. DISCUSSION: Performing a preoperative BRCA test may change the surgical procedure. BRCA tests are beneficial to patients, but the timing of the tests is important. Care must be taken not to force the patient. CONCLUSIONS: Knowing whether the patient is BRCA mutation-positive is extremely important for selecting surgical procedures and treatment methods. BRCA testing should be recommended for patients who are strongly suspected of being positive, but the decision should be the patient's. It is therefore necessary to provide accurate information and engage in a dialogue with the patient, but the medical staff should not pressure the patient to have the test.

Indirect inguinal hernia containing portosystemic shunt vessel: A case report.

BACKGROUND: Inguinal hernia repair is one of the most common general surgical operations worldwide. We present a case of indirect inguinal hernia containing an expanded portosystemic shunt vessel. CASE SUMMARY: We report a 72-year-old man who had a 4 cm × 4 cm swelling in the right inguinal region, which disappeared with light manual pressure. Abdominal-pelvic computed tomography (CT) revealed a right inguinal hernia containing an expanded portosystemic shunt vessel, which had been noted for 7 years due to liver cirrhosis. We performed Lichtenstein's herniorrhaphy and identified the hernia sac as being indirect and the shunt vessel existing in the extraperitoneal cavity through the internal inguinal ring. Then, we found two short branches between the expanded shunt vessel and testicular vein in the middle part of the inguinal canal and cut these branches to allow the shunt vessel to return to the extraperitoneal cavity of the abdomen. The hernia sac was returned as well. We encountered no intraoperative complications. After discharge, groin seroma requiring puncture at the outpatient clinic was observed. CONCLUSION: If an inguinal hernia patient has portal hypertension, ultrasound should be used to determine the contents of the hernia. When atypical vessels are visualized, they may be shunt vessels and additional CT is recommended to ensure the selection of an adequate approach for safe hernia repair.

Primary lymphoma of the breast: A case report and review of the literature.

Primary lymphoma of the breast is a rare disease, accounting for about 0.5% of all primary breast tumors. Often found as a solitary indolent mass, it is difficult to distinguish from breast cancer on imaging and is often diagnosed for the first time based on histological findings. Diffuse large B-cell lymphoma is the most common histological subtype. A 48-year-old woman visited our hospital because of a painless mass in the left breast. Breast cancer was suspected based on the imaging findings. A core needle biopsy was performed, and the invasive ductal carcinoma was diagnosed. Partial mastectomy and sentinel lymph node biopsy were performed. The resected specimen was finally diagnosed as diffuse large B-cell lymphoma based on immunohistochemical staining. The patient was treated with R-CHOP and intrathecal injection of methotrexate. The patient remains alive without recurrence 4 years later. Awareness of primary breast lymphoma is essential for accurate and timely diagnosis and avoidance of unnecessary surgery.

Distal gastric tube resection with vascular preservation for gastric tube cancer: A case report and review of literature.

BACKGROUND: Survival rates in patients with esophageal cancer undergoing esophagectomy have improved, but the prevalence of gastric tube cancer (GTC) has also increased. Total resection of the gastric tube with lymph node dissection is considered a radical treatment, but GTC surgery is more invasive and involves a higher risk of severe complications or death, particularly in elderly patients. CASE SUMMARY: We report an elderly patient with early GTC that had invaded the duodenum who was successfully treated with resection of the distal gastric tube and Roux-en-Y (R-Y) reconstruction. The tumor was a type 0-IIc lesion with ulcer scars surrounding the pyloric ring. Endoscopic submucosal resection was not indicated because the primary lesion was submucosally invasive, was undifferentiated type, surrounded the pyloric ring, and had invaded the duodenum. Resection of distal gastric tube with R-Y reconstruction was safely performed, with preservation of the right gastroepiploic artery (RGEA) and right gastric artery (RGA). CONCLUSION: Distal resection of the gastric tube with preservation of the RGEA and RGA is a good treatment option for elderly patients with cT1bN0 GTC in the lower part of the gastric tube.

Expression levels of FSHR, IGF1R, CYP11al and HSD3ß in cumulus cells can predict in vitro developmental competence of bovine oocytes.

The efficiency of in vitro embryo production technologies would be improved by the development of suitable non-invasive biomarkers that allow the selection of good quality cumulus-oocyte complexes (COCs). The present study used whole, single oocyte culture to investigate whether the expression levels of follicle-stimulating hormone receptor (FSHR), insulin-like factor 1 receptor (IGF1R) and three steroidogenesis-related enzymes (CYP11al, CYP19al and HSD3ß) in cumulus cells reflected the developmental competence of COCs. Cumulus cells were collected from single COCs before maturation culture and relative mRNA levels were assessed using real-time PCR. The analysis indicated that mRNAs for FSHR, IGF1R, CYP11al and HSD3ß were present at higher levels in cumulus cells from COCs that failed to form blastocysts compared with cumulus cells from COCs that formed blastocysts. Moreover, FSHR and IGF1R mRNA levels were positively correlated with those of genes for steroidogenesis-related enzymes. In conclusion, poor developmental competence of COCs was related to higher expression of FSHR, IGF1R, CYP11al and HSD3ß in cumulus cells, which may indicate the advanced differentiation of cumulus cells into granulosa cells.

Local recurrence of breast cancer caused by core needle biopsy: Case report and review of the literature.

INTRODUCTION: Needle tract seeding is the implantation of tumor cells at the site of needle passage during needle biopsy. Histopathological examination of resected specimens after biopsy shows an incidence of 22%-50%. However, reports of actual local recurrence are extremely rare. Here we report such a case. PRESENTATION OF CASE: A 67-year-old woman was diagnosed with ductal carcinoma by histopathology and underwent right mastectomy and sentinel lymph node biopsy. Histopathological examination revealed non-invasive ductal carcinoma. One year after the first operation, a mass was found at the site of the core needle biopsy (CNB) scar near the previous surgical wound on the right chest. Histological examination revealed the tumor as adenocarcinoma, and a skin lesion resection was performed. After surgery, radiation therapy and endocrine therapy were performed. She remains relapse-free as of this writing, 9 months after resection. DISCUSSION: Reports of local recurrence due to needle tract seeding are extremely rare. We found nine cases of mastectomy and seven cases of partial resection performed for the first surgery; six patients received radiation therapy and 10 did not. Histological diagnosis at the time of the first operation was invasive carcinoma in all cases. CONCLUSION: The risk of seeding is high with multiple punctures in CNB, in cases with a short period until surgery, and in mucinous carcinoma. Considering these factors, CNB puncture should preferably be at a site that is included in the resection area during surgery. If not resected, close follow-up is necessary considering the possibility of local recurrence.

Benign phyllodes tumor with hemorrhagic cyst in a 14-year-old girl: A case report.

INTRODUCTION: The incidence of mammary phyllodes tumor is less than 0.3-0.5% of all breast tumors. Only a few pediatric cases have been reported. Here we report a benign phyllodes tumor that occurred in a 14-year-old girl. PRESENTATION OF CASE: A left breast mass with a bleeding cyst of about 9 cm was resected after the tumor had rapidly increased in size over the course of 2 months. Imaging examinations suggested an intracystic tumor. Histopathological examination, however, revealed a benign phyllodes tumor without cytomorphism or mitotic figures. The tumor was accompanied by partial bleeding, which was considered to be the cause of the rapid increase in size of the hemorrhagic cyst. The patient remains under observation, and local recurrence has not occurred as of 3 years after surgery. CONCLUSION: Due to the rarity of the condition in children and adolescents, an individualized, case-based approach is recommended.

A case of encapsulated papillary carcinoma of the breast treated with emergency surgery due to sudden hemorrhage.

INTRODUCTION: Emergency surgery is rare in management of breast cancer. We report a case of encapsulated papillary carcinoma (EPC) of the breast where emergency surgery was performed because of unsuccessful control of hemorrhage. PRESENTATION OF CASE: A 62-year-old woman visited our hospital complaining of sudden bleeding from the right breast. She had been aware of the tumor for a year, but had left it unattended. It had been increasing in size rapidly for a few days before presentation. Computed tomography showed a hypervascular cystic tumor 9 cm in diameter. Bleeding was observed from a skin breach in the right breast, from which a clot was removed and the wound compressed with gauze. We attempted unsuccessfully to control the hemorrhage over the next 3 days, but the patient became anemic, so emergency surgery was performed to control the bleeding. Histopathology revealed the tumor as estrogen receptor- and progesterone receptor-positive EPC, with a human epidermal growth factor receptor 2 score of 0. DISCUSSION: There have been only 5 reports of breast cancer treated with emergency surgery. EPC was previously considered an in situ lesion, but the lack of a myoepithelial layer at the lesion's periphery may represent a low grade or indolent form of invasive carcinoma and prognosis is usually good. However, if bleeding into the cyst occurs and hemostasis is difficult, emergency surgery should be considered. CONCLUSION: Emergency surgery is rare in breast cancer but should always be considered as an option when hemorrhage is not readily controlled.

Bladder metastasis from primary breast cancer: a case report.

BACKGROUND: Breast cancer frequently metastasizes to the bone, lung, and liver. However, metastasis to the bladder is uncommon. Bladder metastasis due to direct infiltration from peripheral organs, such as the colon and rectum, prostate, and cervix, occurs more frequently than metastasis from distant organs, such as the breast. CASE PRESENTATION: We report a case of bladder metastasis identified during treatment for recurrent breast cancer. Fifteen years after her initial surgery, a known breast cancer patient complained of a left lower abdominal pain, anuria, and body swelling. Computed tomography imaging revealed an irregular thickening of the left bladder wall, left hydronephrosis, and hydroureter. A bladder metastasis from breast cancer was diagnosed based on a histological examination of a cystoscopic biopsy specimen. She is currently receiving chemotherapy with eribulin mesylate. CONCLUSIONS: Routine screening of the lower urinary tract is not necessary for all patients, but women with a history of breast cancer presenting with urinary symptoms should undergo a thorough examination of the urinary tract.

Duodenal infusion of fatty acids differentially affects plasma glucagon-like peptide-1 and ghrelin concentrations in sheep.

The aim of this study was to investigate how intraduodenal infusions of fatty acids (FA) affect appetite-related gut peptides such as glucagon-like peptide-1 (GLP-1) and ghrelin in sheep. We hypothesized that these peptides can be highly reactive to unsaturated long-chain FA, because they are well known to decrease dry matter intake (DMI). Four ewes were fitted with a duodenal cannula and a jugular vein catheter for a 6-h duodenal infusion of the 9 FA (C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, and C18:3) and water (control). The concentration of each FA was 1.6 g per metabolic body weight (BW), approximately corresponding to the amount of supplemented fat in a standard dairy cow diet. Each infusion was separated by at least 2 d. During the infusion period, blood samples were collected periodically to determine changes in plasma GLP-1, ghrelin, and metabolite concentrations. Duodenal infusions of C18:1, C18:2, and C18:3 led to higher plasma GLP-1 (P < 0.05) and lower glucose (P < 0.05) than control. Plasma ghrelin concentrations were greater in C18:1 and C18:3 infusions than control (P < 0.05). Plasma ketone bodies were higher in C8:0 and C10:0 infusions (P < 0.05), but plasma triglyceride concentrations were lower in C8:0, C10:0, C12:0, and C16:0 infusions (P < 0.05) than control. Fatty acid infusions except for C18:3 led to higher plasma NEFA concentrations than control (P < 0.05). These results confirmed that the hypophagic effect of dietary unsaturated long-chain FA is mediated by GLP-1 (an anorexigenic effect) secretion. However, we also observed higher plasma ghrelin (an orexigenic effect) partially by unsaturated long-chain FA. Thus, the gut peptide secretions when ruminant animals ingest FA supplements would complexly affect satiety and further studies are needed to determine their each impact on DMI.

Effects of insulin-like growth factor-1 on the in vitro maturation of canine oocytes.

The maturation rate of canine oocytes during in vitro maturation (IVM) needs to be improved. The present study was designed to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the IVM of canine oocytes. Ovaries were obtained by ovariohysterectomy and were sliced to release cumulus-oocyte complexes (COCs). In Experiment 1, the effects of different concentrations of IGF-1 on the nuclear maturation of oocytes was investigated. The COCs were cultured in a modified medium (mTCM199) with IGF-1 (0, 0.5, 5, 10, and 50 µg/ml). At the end of the 48 h culture, oocytes were fixed and stained to evaluate their nuclear stage. Supplementation with 50 µg/ml IGF-1 induced a significantly higher metaphase II (MII) rate (P < 0.05) compared to the 0 and 0.5 µg/ml IGF-1 groups. In Experiment 2, the expression levels of insulin receptor (INSR), IGF-1 receptor (IGF-1R), and IGF-2 receptor (IGF-2R) genes, localized to canine oocytes and cumulus cells, were investigated before and after IVM. The expression level of IGF-1R in cumulus cells after IVM was higher than that before IVM (P < 0.05). In Experiment 3, it was investigated whether an inhibitor of PTEN (phosphatase and tensin homolog), bpV, affects the nuclear maturation of oocytes. Regardless of bpV supplementation at a concentration of 0.2 to 200 µmol/l, there was no significant difference in the proportion of oocytes that reached the MII stage. These results indicated that IGF-1 has a favorable effect on the IVM of canine oocytes, possibly through the stimulation of the Ras/MAPK pathway via IGF-1R expressed in cumulus cells.

Histochemical properties of bovine and ovine mammary glands during fetal development.

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination.

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.