Polymorphism in promoter region of growth hormone receptor is associated with potential production capacity of insulin-like growth factor-1 in pre-pubertal Holstein heifers.

Insulin-like growth factor-1 (IGF-1) is one of the important factors for growth, milk production and reproductive functions and mainly released from the liver in response to growth hormone (GH) via GH receptor (GHR) in cattle. Recently, some single nucleotide polymorphisms (SNPs) were identified in the bovine GHR gene. Some GHR-SNPs were shown to be related to plasma IGF-1 concentration in cattle. Hence, the capacity to IGF-1 production in the liver might be affected by GHR-SNP and associated with performance in the future. This study examined whether GHR-SNP is associated with IGF-1 production in the liver of pre-pubertal heifers. In 71 Holstein calves, blood samples for genomic DNA extraction were obtained immediately after birth. To genotype the GHR-SNPs in the promoter region, polymerase chain reaction (PCR) products were digested with restriction enzyme NsiI (cutting sites: AA, AG and GG). All heifers at 4 months of age were intramuscularly injected with 0.4 mg oestradiol benzoate. Blood samples were obtained from the jugular vein just before (0 h) and 24 h after injection. The number of AA, AG and GG at the NsiI site was 0, 17 and 54 respectively. In AG and GG, plasma GH concentrations were higher pre-injection than 24 h post-injection (p < 0.01). Moreover, plasma GH concentrations in AG post-injection were higher than in GG (p < 0.05). In contrast, the GG genotype exhibited higher plasma IGF-1 concentrations in pre-injection than post-injection (p < 0.01), although oestradiol did not change IGF-1 concentration in the AG genotype. We conclude that the GG polymorphism in the promoter region of GHR is associated with a higher potential capacity of IGF-1 production in the liver of cattle.

BRCAness is beneficial for indicating triple negative breast cancer patients resistant to taxane.

AIM: Triple negative breast cancer (TNBC) is a heterogeneous disease and is associated with the cancer stem cell (CSC), basal-like, and BRCA1 function deficient (BRCAness) subtypes. We examined these 3 subtypes in TNBC and compared their chemosensitivity against anthracycline or taxane with a special attention to BRCAness. METHODS: Sixty-six TNBC cases were obtained from a randomized phase II trial comparing TCx6 (TC6) with FEC-Docetaxel (FEC-D) as neoadjuvant chemotherapy. The core needle specimens before chemotherapy were used for subtyping. The basal-like and CSC subtypes were identified by immunohistochemistry; CK5/6 and EGFR staining for the basal-like subtype and ALDH1 staining for the CSC subtype. The BRCAness subtype was examined by Multiplex Ligation-dependent Probe Amplification (MLPA). Correlations between subgroups and pCR rates according to each regimen and subtype were examined. RESULTS: The basal-like and BRCAness subtypes were significantly associated (p = 0.010) with the other subtypes, but not the CSC subtype. The pCR rates were higher with FEC-D than with TC6 in the basal-like (54.5% vs 14.3%, p = 0.081) and BRCAness (56.2% vs 16.7%, p = 0.030) subtypes. Both were not effective in the CSC subtype (18.2% vs 11.8%, p = 1.00). CONCLUSION: BRCAness identified by MLPA was practically useful for treatment selection for avoiding taxane. ALDH1 may be considered as a marker for the CSC subtype requiring novel agents.

A new method for characterizing hand dysfunction in cervical spondylotic myelopathy: a preliminary study.

STUDY DESIGN: A case-control investigation. OBJECTIVES: The objective of this study was to quantitatively study impaired ability to appropriately adjust pinch strength while holding a small object in patients with cervical spondylotic myelopathy (CSM). SETTING: Kochi Medical School Hospital, Japan. METHODS: The subjects consisted of 19 CSM patients who had frequent episodes of failing to grasp and hold small objects in their daily life (Group A), 13 CSM patients who did not experience such episodes (Group B) and 16 healthy subjects (Control Group). We continuously measured the dynamic internal pressure of a pneumatic rubber object called a blower pinched by the subject, following two different sets of instructions: (1) pinching with eyes open and with the minimal strength required to prevent dropping; and (2) maintaining a constant pinch strength at given levels with eyes closed. RESULTS: Compared with the other two groups, Group A subjects used a significantly (P<0.01) greater pinch strength to avoid dropping the blower held with eyes open and showed a significantly (P<0.01) greater deviation in pinch strength from the baseline values with eyes closed. These tendencies in Group A showed a significant correlation with the tactile perception threshold of the digits (P<0.01) but not with impairment of rapid repetitive movements of the digits that reflects spasticity. CONCLUSION: Our technique applied to CSM patients helps assess functional integrity primarily, if not exclusively, of the fasciculus cuneatus mediating the feedback signals from proprioceptive and cutaneous receptors in the digits, which are otherwise difficult to evaluate quantitatively.

Identifying alterations in metabolic profiles of dairy cows over the past two decades in Japan using principal component analysis.

The aim of this study was to identify the metabolic changes that have occurred in Holstein-Friesian cows in Japan over the past 2 decades, based on long-term metabolic profiles. From 1987 to 2004, metabolic profile tests were conducted in 1,700 dairy herds containing ~50,000 cows. The cows were divided into 5 lactation stage groups: early (calving to 49 d in milk, DIM), peak (50-109 DIM), mid (110-209 DIM), and late (210 to dry-off) lactation and the dry period. Principal component analysis was applied to the metabolic profiles at each lactation stage separately to investigate changes in metabolic profiles across the multiyear periods 1987-1992, 1993-1998, and 1999-2004. We determined that cows have probably experienced increasing negative energy balance, energy deficiency, and liver dysfunction during the past 2 decades in Japan.

A prospective feasibility study of sentinel node biopsy by modified Indigocarmine blue dye methods after neoadjuvant chemotherapy for breast cancer.

BACKGROUND: Although sentinel lymph node biopsy (SLNB) is a standard staging method for assessing nodal status of breast cancer patients, SLNB after neoadjuvant chemotherapy (NAC) remains controversial. The aim of this study was to validate the practicality and accuracy of SLNB by our modified Indigocarmine blue dye methods following NAC. METHODS: One hundred consecutive cases with breast cancers treated by NAC were enrolled in this study. After NAC, all patients underwent SLNB performed by our modified Indigocarmine blue dye methods without radioisotope, followed by back-up axillary lymph node dissection (ALND). RESULTS: Sentinel nodes (SNs) were identified in 94 cases (identification rate, 94%); the accuracy was 94.7% (89/94 cases); and the false negative rate (FNR) 13.5% (5/37 cases). For cases with vs. without clinically evident metastatic nodes before NAC, the identification rate was 92.4% (61/66 cases) vs. 97.1% (33/34 cases); the accuracy 91.8% (56/61 cases) vs. 97.0% (32/33 cases) and the FNR 16.1% (5/31 cases) vs. 0% (0/6 case), respectively. There were six patients without identified SNs, three of them had metastatic nodes. False negatives occurred in five cases; in four, fewer than two sentinel nodes had been removed. CONCLUSION: Following NAC, the accuracy of SLNB by modified Indigocarmine blue dye methods is adequate compared with other tracers. In patients in whom no SNs have been identified, lymphatic metastasis is likely and therefore ALND is recommended. For patients with cN0 prior to NAC, SLNB by modified Indigocarmine blue dye methods is clinically feasible, though controversial for patients with positive nodes.

Outcomes of immediate perforator flap reconstruction after skin-sparing mastectomy following neoadjuvant chemotherapy.

BACKGROUND: The impact of neoadjuvant chemotherapy (NACT) on immediate free flap breast reconstruction remains controversial. Furthermore, the oncological outcomes of immediate free flap breast reconstruction after skin-sparing mastectomy (SSM) following NACT remain unclear. This study aimed to investigate the surgical complications and oncological outcomes of immediate perforator flap reconstruction after SSM following NACT. METHODS: A total of 201 consecutive patients with indications for immediate perforator flap reconstruction after SSM were included between 2004 and 2012. Surgical and oncological outcomes were compared between patients with and without NACT. RESULTS: There were 38 patients in the NACT group and 163 in the non-NACT control group. The median age of the NACT group was 39.5 years, which was significantly younger than the control group (43.0 years; P < 0.05). Patients in the NACT group also had more advanced and aggressive disease (P < 0.05). There was no significant difference in the frequency of surgical complications between the groups, no difference in the type of complications, and no significant difference in the frequencies of major and minor complications. No patients in the NACT group had delayed adjuvant therapy. Eight patients (4%) developed recurrences, with a median follow-up time of 3.0 years. Local recurrences occurred in three control patients but no patients in the NACT group. CONCLUSION: NACT does not affect short-term or interim outcomes after immediate perforator flap reconstruction and may thus represent a safe and practical treatment option for the multidisciplinary treatment of breast cancer.

Imaging findings of necrotizing sialometaplasia of the parotid gland: case report and literature review.

Although necrotizing sialometaplasia (NS) of the parotid gland is rare and occasionally presents as a lesion that mimics a malignant tumour, imaging findings in cases of NS have been rarely reported. We describe here a case of NS in which there was an increasing lesion manifesting overnight on the parotid gland in an 83-year-old male. We also investigated the use of pre-operative imaging based on previous reports and discuss the importance of these images in helping to guard against overzealous treatment. It is critically important to closely examine whether there are aspects of NS, such as the present case, in pre-operative MRI findings that can be useful in proper diagnosis and treatment.

Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra.

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-alpha mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P<0.05). In bitches with pyometra, endometrial TGF-alpha and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P<0.05). In normal bitches, positive immunohistochemical staining for TGF-alpha, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-alpha and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-alpha by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.

Ovarian stimulation with follicle-stimulating hormone under increasing or minimal concentration of progesterone in dairy cows.

The objective of this study was to investigate the effect of the presence or absence of Corpus luteum (CL) on the follicular population during superstimulation in dairy cows (Holstein-Friesian cattle). Animals were divided into two groups as follows: (1) Growing CL group (G1): Cows (n=7) received a total dose of 28 Armour units (AU) follicle-stimulating hormone (FSH) through the first 4 d (twice daily) after spontaneous ovulation (Day 0). (2) CL Absence group (G2): Cows (n=10) received prostaglandin F(2alpha) (PGF(2alpha)) at 9 or 10 d after ovulation. After 36h, all the follicles (larger than 5mm) were aspirated (Day 0). The FSH treatment started 24h after aspiration and continued for 4 d. The number of small (3 to <5mm), medium (5 to <8mm), and large (> or = 8mm) follicles was examined on Days 1, 3, and 5 in all groups. Blood samples were collected daily for 5 d, and progesterone (P(4)), estradiol (E(2)), insulin-like growth factor-1 (IGF-1), and growth hormone (GH) in plasma were measured by enzyme immunoassays. The results showed that in G1, the P(4) level increased gradually from 0.5 ng/mL at Day 1 to 2 ng/mL at Day 5, whereas in G2, the P(4) level was completely below 0.5 ng/mL. All cows of the G2 group showed an increase of E(2) at Day 3 or Day 4 followed by an increase of IGF-1 within 24h, while GH increased concomitantly with the E(2) increase in 8 of 10 trials. On the other hand, cows of the G1 group showed neither E(2) nor IGF-1 increase. Moreover, at the end of the treatment, the number of follicles in the G2 group was significantly increased compared with that of the G1 group (22.8+/-2.0 vs. 11.6+/-2.0). In conclusion, low P(4) level during FSH treatment enhanced multiple follicular growth and E(2) secretion, which was followed by increase of IGF-1 and GH. Therefore, the absence of the CL may play a critical role in the superovulation response by controlling the number of growing follicles.

Effect of ß-carotene supply during close-up dry period on the onset of first postpartum luteal activity in dairy cows.

The aim of this study was to examine the effect of ß-carotene supply during the close-up dry period on the onset of first postpartum luteal activity in dairy cows. Twelve cows were supplied with 2000 mg of ß-carotene (20 g Rovimix(®) ß-Carotene containing 10% ß-carotene; DSM Nutrition Japan K.K., Tokyo, Japan) by oral administration daily from day 21 before expected calving date to parturition. Fourteen cows (control) did not receive ß-carotene supplementation. Blood samples were obtained on days 21, 14 and 7 before expected calving date and on days 1, 7, 14, 21 postpartum. When the plasma progesterone concentration exceeded 1 ng/ml by day 21 postpartum, luteal activity was assumed to have been initiated. The result showed that serum ß-carotene concentrations in the ß-carotene cows were higher than in the control cows during the experimental period (p < 0.01). The number of cows with the onset of luteal activity by day 21 postpartum was 9/12 in the ß-carotene cows and 4/14 in the control cows (p < 0.05). Retinol, certain metabolic parameters and metabolic hormones concentrations did not differ between ß-carotene and control cows. In addition, serum retinol concentration in ß-carotene cows without luteal activity was lower than in ß-carotene cows with luteal activity (p < 0.05), and serum gamma-glutamyl transpeptidase concentration in ß-carotene cows with luteal activity (p < 0.05) and control cows without luteal activity (p < 0.01) was higher than in control cows with luteal activity. In conclusion, ß-carotene supply during the close-up dry period may support the onset of luteal activity during early lactation in dairy cows.

Canine oocyte maturation in culture: significance of estrogen and EGF receptor gene expression in cumulus cells.

We examined the role of cumulus cells regarding in vitro maturation of canine oocytes, and investigated estrogen and epidermal growth factor (EGF) receptor gene expression and action on nuclear maturation. Canine cumulus-oocyte complexes (COC) were collected from anestrous and diestrous bitches; only COC with vitelline diameter >100 microm were used. In Experiment 1, expression of estrogen receptor (ER) alpha, ERbeta and EGF-receptor (EGF-R) were determined by reverse transcription-polymerase chain reaction (RT-PCR), using mRNA from the oocyte or cumulus cell. Transcripts for the ERbeta and EGF-R were detected in oocytes and cumulus cells, but no message was detected for ERalpha. In Experiment 2, intact COC and the denuded oocytes were cultured in TCM199 medium supplemented with various concentrations of estradiol-17beta (E(2); 0-10 microg/mL) or EGF (0-100 ng/mL) for 72 h; nuclear maturation was then evaluated. In oocytes cultured within intact COC, the rate of germinal vesicle breakdown (GVBD) was higher in the 1 microg/mL E(2) supplemented group (P<0.05), and the rate of metaphase I (MI) was higher in the 10 ng/mL EGF supplemented group, than in the non-supplemented group (P<0.05). However, supplementation of E(2) or EGF to denuded oocytes failed to promote nuclear maturation. In Experiment 3, intact COC were cultured in TCM199 supplemented with 1 microg/mL E(2), 10 ng/mL EGF, and 10% fetal bovine serum (FBS) for 72 h, and nuclear maturation was evaluated. There was no significant difference in the rate of metaphase II (MII) between the medium only, E(2)+EGF, and FBS supplement groups. When E(2) and EGF in combination with FBS were supplemented, the rate of MII was higher than in other groups (P<0.05). We inferred that cumulus cells were involved in mediating the stimulatory effects of E(2) and EGF on nuclear maturation of canine oocytes, and that E(2) and EGF in combination with FBS promoted the completion of oocyte meiotic maturation.

Relationship between plasma beta-carotene concentrations during the peripartum period and ovulation in the first follicular wave postpartum in dairy cows.

Beta-carotene functions independently of vitamin A in the reproductive performance of dairy cows. The concentrations of beta-carotene in plasma decrease during the dry period, and reach a nadir in about the first week postpartum. This coincides with a negative energy balance, which affects the onset of the first ovulation in early postpartum cows. Thus, we hypothesised that plasma beta-carotene concentrations during the peripartum period may affect ovulation in the first follicular wave postpartum in dairy cows. The aim of the present study was to investigate changes in the profiles of plasma beta-carotene concentrations during the peripartum period in ovulatory and anovulatory cows during the first follicular wave postpartum. We used 22 multiparous Holstein cows, which were fed a total mixed ration consisting of grass, corn silage and concentrate, and collected blood samples for beta-carotene and progesterone analysis from week 3 prepartum to week 3 postpartum when the period of day 0-6 after parturition was regarded as the parturient week (week 0). The first ovulation was confirmed using the profile of plasma progesterone concentrations and colour Doppler ultrasound. Thirteen cows ovulated during the first postpartum follicular wave. Parity, the dry-off period, calving interval, mastitis episodes, and actual 305 days' milk yield during the previous lactation, and milk composition in the last month during the previous lactation in this study did not differ between ovulatory and anovulatory cows. Differences in the plasma beta-carotene profile were observed between ovulatory and anovulatory cows. Plasma beta-carotene concentrations at week 3 prepartum were greater in ovulatory cows (2.97+/-0.24 mg/L) than in anovulatory cows (1.53+/-0.14 mg/L; P<0.001), after that its concentrations in ovulatory cows decreased and reached the lowest level at week 1 postpartum, although its concentrations in anovulatory cows remained unchanged. No differences in plasma beta-carotene concentrations between the two groups were observed postpartum. The present study indicates for the first time that the lower beta-carotene concentrations in plasma during the prepartum period is associated with anovulation during the first follicular wave postpartum.

Postoperative pain status after intraoperative systemic dexmedetomidine and epidural neostigmine in patients undergoing lower abdominal surgery.

BACKGROUND AND OBJECTIVES: To determine whether intraoperative systemic dexmedetomidine improves postoperative pain and interacts with epidural neostigmine to produce analgesic effects. METHODS: Sixty patients undergoing gynaecological surgery were randomly divided into four groups to receive epidural neostigmine and/or systemic dexmedetomidine: control (Group C), epidural neostigmine (Group N), systemic dexmedetomidine (Group D) and co-administered neostigmine and dexmedetomidine (Group ND). Epidural neostigmine (0.3 mg) was administered with 10 mL of 0.75% ropivacaine before the induction of general anaesthesia. Systemic dexmedetomidine (loading dose of 1 mug kg-1 over 10 min followed by 0.4 mug kg-1 h-1) was infused after the induction of general anaesthesia and continued until the end of surgery. The pain status of patients was assessed using the visual analogue scale at 2, 4, 6, 24 and 72 h postoperatively. RESULTS: Intraoperative systemic dexmedetomidine alone did not reduce postoperative pain scores. However, co-administered neostigmine and dexmedetomidine significantly decreased scores at 24 and 72 h (Group C: 3.0 [1.0-5.8] and 2.0 [0.3-3.0]; Group N: 1.5 [0.3-3.4] and 0 [0-1.3]; Group D: 3.5 [0-5.0] and 0 [0-1.4]; and Group ND: 0 [0-1.0]* and 0 [0-0]; median [interquartile range] *P = 0.0031, P = 0.0045 compared with Group C). CONCLUSIONS: The intraoperative systemic infusion of dexmedetomidine alone at doses causing sedation does not result in postoperative analgesic effects. However, the co-administration of systemic dexmedetomidine and epidural neostigmine at higher doses may be a useful method to improve postoperative pain although side-effects have to be evaluated.

Peripheral administration of kisspeptin-10 increases plasma concentrations of GH as well as LH in prepubertal Holstein heifers.

This study was conducted to estimate the effects of kisspeptin-10 on blood concentrations of LH and GH in prepubertal dairy heifers. Heifers received a single injection of 1 mg kisspeptin-10 (n=5) or saline (n=5) intravenously, and serial blood samples were collected at 15-min intervals to analyze the response curves of both LH and GH after injection. Peak-shaped responses were observed for concentrations of LH and GH, and the peaks were observed at 27+/-3 and 75+/-9 min, respectively, after injection, only in heifers injected with kisspeptin-10. These data suggest various possible important links among kisspeptin, the reproductive axis, and also the somatotropic axis in prepubertal Holstein heifers.

Short communication: A positive relationship between the first ovulation postpartum and the increasing ratio of milk yield in the first part of lactation in dairy cows.

The aim of the present study was to examine the relationship between characteristics of the lactation curve, on the basis of daily milk yield, and ovulation within 3 wk postpartum as an indicator of early return to luteal activity in dairy cows. Lactation records from 46 lactating Holstein cows between calving and 305 d postpartum were studied. Milk samples were collected twice weekly between d 7 and 100 for later determination of progesterone concentrations. Occurrence of an early first ovulation was determined by an increase in milk progesterone by 3 wk after calving. Milk yield was recorded daily until 305 d postpartum, and average yield was calculated weekly. The lactation curve was characterized by 8 indices on the basis of the weekly average of milk yield as follows: a) first-week milk yield; b) peak milk yield; c) actual 305-d milk yield; d) peak week; e) difference in milk yield between the first week and peak week; f) difference in milk yield between the peak week and last week (43rd week postpartum); g) ratio of increase in milk yield between wk 1 and the week of peak yield; and h) ratio of decline in milk yield between the week of peak yield and the last week. Indices g and h were calculated as linear. The number of cows having ovulated by 3 wk postpartum was 22 (47.8%). The resumption of ovarian cycles with normal luteal phases occurred earlier in ovular cows than in anovular cows (32.0 d vs. 57.1 d). Although total milk yield did not differ between ovular and anovular cows, the ratio of increase in milk yield from the first week to the peak week (index g) in ovular cows was smaller compared with that of anovular cows (1.71 vs. 2.54). In addition, the ratio of increase in milk yield from the first week to the third week postpartum was greater in anovular cows by 3 wk postpartum (ovular = 1.43 +/- 0.23 vs. anovular = 2.32 +/- 0.29). In conclusion, the present study demonstrates that a greater increasing ratio of milk yield during early lactation may delay resumption of ovarian cycles after parturition. Therefore, this study is the first to demonstrate statistically that a smaller increasing ratio of milk yield (index g) during early lactation may have a beneficial effect on the first ovulation by 3 wk postpartum.

Preventive effects of CIDR-based protocols on premature ovulation before timed-AI in Ovsynch in cycling beef cows.

Ovsynch is a program developed to synchronize ovulation for timed breeding. In this paper, the authors investigate whether controlled internal drug release (CIDR)-based protocols prevent premature ovulation before timed-artificial insemination (AI) when Ovsynch is started a few days before luteolysis in cycling beef cows. Nine beef cows at 16 days after oestrus were treated with (1) Ovsynch, i.e. gonadotropin releasing hormone (GnRH) analogue on day 0, prostaglandin (PG) F(2alpha) analogue on day 7 and GnRH analogue on day 9 with timed-AI on day 10, (n=3); (2) Ovsynch+CIDR (Ovsynch protocol plus a CIDR for 7 days from day 0, n=3), or (3) oestradiol benzoate (OB)+CIDR+GnRH (OB on day 0 in lieu of the first GnRH treatment, followed by the Ovsynch+CIDR protocol, n=3). In the Ovsynch group (1) plasma progesterone concentrations fell below 0.5 ng/mL earlier (day 5) than in both CIDR-treated groups (2) and (3), where this occurred on day 8. Plasma oestradiol-17beta concentrations peaked on day 8 in the Ovsynch group and on day 9 in both CIDR-treated groups. The dominant follicle ovulated on day 10 in the Ovsynch group and on day 11 in both CIDR-treated groups. Thus, both CIDR-based protocols prevented premature ovulation before timed-AI in Ovsynch when the protocol was started a few days before luteolysis. This reflects the fact that progesterone levels remained high until the beef cattle were treated with PGF(2alpha).

Lactoferrin expression in the canine uterus during the estrous cycle and with pyometra.

The expression of lactoferrin, a non-specific antimicrobial defence, in the canine uterus during the normal estrous cycle and in bitches with pyometra was examined. Using polymerase chain reaction analysis, lactoferrin gene transcripts were detected in the endometrium at all stages of the estrous cycle, with the highest levels in estrus. In normal bitches, endometrial lactoferrin mRNA increased from proestrus to estrus (P<0.05). Thereafter, it dramatically decreased from estrus to Day 10 of diestrus (P<0.05), and stayed low at Day 35 of diestrus and anestrus; this was consistent with blood estrogen concentrations. Levels of lactoferrin mRNA were higher in bitches with pyometra than in normal diestrus (P<0.05). With immunohistochemistry, distinct staining of lactoferrin was detected in the luminal and glandular epithelial cells of the endometrium at proestrus and estrus, but little staining was detected at Day 10 of diestrus. At Day 35 of diestrus and anestrus, a partial and weak reaction was present in the same region. In bitches with pyometra, the glandular epithelial cells and many cells in the uterine stroma were strongly stained. Staining cells in the stroma were morphologically similar to neutrophils. No lactoferrin staining was seen in the uterine stromal cells or myometrium in any section. These results suggest that, in the canine uterus, lactoferrin expression is related to the blood concentration of estrogen, and that the dramatic reduction in lactoferrin observed at the early stage of diestrus may impair antimicrobial defense. Also, enhanced expression of lactoferrin mRNA in the endometrium with pyometra may be associated with neutrophil invasion into the uterus to combat the infection.

Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes.

We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 microm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199+10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 microg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs-Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO2 in air at 38.5 degrees C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control (P<0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group (P<0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.