Effects of human chorionic gonadotropin and intravaginal progesterone device treatment after artificial inseminations on the reproductive performance of normal and repeat breeder lactating dairy cows.

We examined the effects of human chorionic gonadotropin (hCG) treatment on Day 5 (Day 0 = day of artificial insemination: AI) and intravaginal progesterone device (IVPD) treatment from Day 5 to 19 on the conception and detection rates of return to estrus (re-estrus) in lactating dairy cows. A total of 306 cows from a commercial dairy farm were divided into the following three groups on Day 5: non-treatment group (n = 128), untreated; hCG group (n = 71), 3,000 IU hCG was administered (intramuscularly); IVPD group (n = 107), IVPD was inserted into the vagina from Day 5 to 19. Re-estrus detection was performed up to Day 25. Pregnancy was diagnosed by rectal palpation between Day 50 and 60. There was an interaction between treatment and AI number (P < 0.01) on the conception rate of first-AI. For cows with more than three AIs, the IVPD treatment (66.7%) was more effective than the non-treatment (23.1%) (P < 0.05). The re-estrus detection rate was significantly (P < 0.05) higher in the IVPD group (60.7%) than that in the non-treatment group (41.4%) and tended (P < 0.1) to be higher than that in the hCG group (37.8%). Our results suggested that the conception rate can be improved by IVPD treatment, especially in cows with more than three AIs. In addition, IVPD treatment can induce higher estrus expression up to 25 days after AI in non-pregnant cows.

Evidence that the dominant follicle of the first wave is more active than that of the second wave in terms of its growth rate, blood flow supply and steroidogenic capacity in cows.

To clarify the different characteristics of the dominant follicle (DF), the DF in first ovarian follicular wave (DF-1) after spontaneous ovulation and DF in second follicular wave (DF-2) and after induced ovulation of the first-wave DF by GnRH were examined in non-lactating Holstein cows. Follicular maturation of DF-1 and DF-2 were induced by PGF2α and GnRH treatment on Day 6 and 8 (Day 0=Day of follicular wave emergence), respectively. Follicular growth and blood flow (BF) in the follicular wall of DF-1 and DF-2 were examined. To analyze sex steroids in follicular fluid (FF) and amount of mRNA in granulosa cells, DF-1 and DF-2 were aspirated on Day 8 or 9 in different estrous cycle. Diameter in DF-1 was larger than DF-2 on Day 8 and 9. From Day 8 to 9, BF area (BFA) and percentage of the follicular wall with BF, which represents the degree of distribution of BF, increased in DF-1 but not in DF-2. BFA per length of follicle circumference with BF, which represents the thickness of BF, was not different between DF-1 and DF-2. Concentrations of 17ß-estradiol (E2) in plasma, E2 and androstendione in FF and amounts of LH receptor mRNA were greater in the DF-1 on Day 8. Gene expression for steroidogenesis, prostaglandin synthesis and angiogenesis did not differ between DF-1 and DF-2. These results indicated that DF-1 were more active than DF-2 in growth, BF supply and steroidogenesis. The greater BFA observed in the DF-1 may be derived from as a result of the greater vascularity in the follicular wall.

Basal levels and GnRH-induced responses of peripheral testosterone and estrogen in Holstein bulls with poor semen quality.

The present study investigated the basal levels and GnRH-induced responses of peripheral testosterone and estrogen in Holstein bulls with poor semen quality. On the basis of semen parameters, bulls (n=5) having poor semen quality were selected as experimental bulls, and good semen quality bulls (n=4) were used as control bulls. Both groups were treated intramuscularly once with GnRH (250 µg of fertirelin acetate). Blood samples were collected at -1 day (d), -30 min and 0 h (treatment) followed by every 30 min for 5 h and 1, 3 and 5 d post-GnRH treatment (PGT), and LH, testosterone and estradiol-17ß (E(2)) concentrations were measured. The pretreatment concentrations were used as basal levels. The percentage increments based on the 0-h levels were calculated per bull for each sampling time until 5 h PGT, and differences were compared between the experimental and control groups. The PGT concentrations of testosterone and basal and PGT concentrations of E(2) were significantly lower in the experimental group. The testosterone increment in the experimental group was delayed and significantly lower from 1 to 5 h PGT than those in the control group. It can be suggested that bulls with poor semen quality have delayed and lower GnRH-induced testosterone response and may also have lower estrogen levels.

Effects of scrotal insulation and pathological lesions on alpha-Smooth Muscle Actin (SMA) and vimentin in the bull testes.

The expression patterns of alpha-smooth muscle actin (SMA) and vimentin in the bovine testis indicate the maturation status of seminiferous tubules. The present study demonstrates the effects of impaired spermatogenesis resulting from increased testicular temperature after scrotal insulation and pathological lesions in subfertile bull testes on the changes in immunohistochemical expression of alpha-SMA and vimentin. Scrotal insulation induced degenerative changes of seminiferous tubules, and subfertile bull testes demonstrated characteristic mixed atrophied lesions; dysplastic lesions were seen in one bull. The increased intensity of peritubular alpha-SMA in the dysplastic area was distinct and indicated shrinkage of the seminiferous tubules. The mixed atrophied lesions revealed unaltered expression of peritubular alpha-SMA. However, considerable distortion was observed in the expression of alpha-SMA in severely degenerated tubules after insulation, which may indicate the heat sensitivity of peritubular alpha-SMA or its relation with spermatogenic activity under sudden heat stress. The vimentin expression pattern in the degenerated tubules of post-insulated testes was unaltered. However, the Sertoli cell-only tubules of mixed atrophied subfertile bull testes were characterized by an increase in vimentin of strong intensity resembling that in the transforming pattern, which may indicate the reversion of Sertoli cell maturity in such cases.

Significance of imaging features of alveolar echinococcosis in studies on nonhuman primates.

In this study, we report the imaging findings in two Japanese monkeys (Macaca fuscata) diagnosed with alveolar echinococcosis. Both monkeys were treated with albendazole for 10 years, without surgery. Radiography, computed tomography, and contrast-enhanced ultrasonography were performed under general anesthesia. This is the first report on contrast-enhanced ultrasonographic imaging for alveolar echinococcosis wherein perflubutane was used as the contrast medium. The findings of the imaging analyses were similar to those reported for alveolar echinococcosis in humans, such as snowflake sign and worm-eaten sign. In addition, the serology correlated well with the imaging data in these two monkeys. Therefore, we propose that the imaging findings of alveolar echinococcosis in nonhuman primates may be used to accumulate data on this condition in human alveolar echinococcosis.

Quantitative evaluation of hip joint laxity in 22 Border Collies using computed tomography.

The purpose of this study was to obtain the computed tomography (CT) data for the hip joints of 22 Border Collies. The dorsolateral subluxation (DLS) score, lateral center edge angle (LCEA), dorsal acetabular rim angle (DARA) and center distance (CD) index were measured on the CT images in a weight-bearing position. Radiographic Norberg angle (NA) was also measured. The mean values were 45.7 +/- 10.2% for DLS score, 85.9 degrees +/- 10.3 degrees for LCEA, 18.5 degrees +/- 7.3 degrees for DARA, 0.40 +/- 0.17 for CD index and 102.7 degrees +/- 6.9 degrees for NA. Since the DLS score and LCEA showed strong correlation, combined use of these parameters might improve diagnostic accuracy. We consider CT evaluation in a weight-bearing position to be a useful method for multidirectional evaluation of hips.

Analysis of blood flow in a third ventricular ependymoma and an olfactory bulb meningioma by using perfusion computed tomography.

Brain perfusion computed tomography (CT) scanning was performed in a mongrel dog and a golden retriever that were diagnosed with third ventricular tumor and olfactory bulb tumor, respectively, by contrast-enhanced CT. The tumors were pathologically diagnosed as ependymoma and meningioma, respectively. Perfusion CT results revealed that the ependymoma in this study had a lower blood flow, higher blood volume, and greater transit time of blood than the adjacent brain tissue. Further, the meningioma in this study had a higher blood flow, higher blood volume, and greater transit time of blood than the adjacent brain tissue. Perfusion CT can potentially be used for the grading of brain tumors and narrowing differential diagnosis, provided the perfusion CT data of animals are accumulated.

Relationships among age, body weight, scrotal circumference, semen quality and peripheral testosterone and estradiol concentrations in pubertal and postpubertal Holstein bulls.

In the present study, the correlations among age, body weight, scrotal circumference (SC), semen quality and peripheral testosterone and estradiol-17beta (E(2)) concentrations were investigated in pubertal (n=5) and postpubertal (n=7) groups of Holstein bulls over a 6 week period. There were significant positive correlations (P<0.01) among age, body weight and SC in both groups, and similar significant correlations between sperm motility and SC in pubertal bulls (P<0.01) and between sperm concentration and SC in postpubertal bulls (P<0.05). The sperm motility after collection (P<0.05) and after freezing and thawing (P<0.01) of the postpubertal bulls correlated positively with the E(2) concentration. Estrogen may be important for the function of postpubertal bull testes, in which it may regulate spermatozoa motility in vivo.

Induction of ovulation with GnRH and PGF(2 alpha) at two different stages during the early postpartum period in dairy cows: ovarian response and changes in hormone concentrations.

The aims of this study were 1) to determine whether dairy cows can be induced to ovulate by the treatment with gonadotropin releasing hormone (GnRH) followed by prostaglandin F(2 alpha) (PGF(2 alpha)) during the early postpartum period and 2) to describe their ovarian and hormonal responses according to ovarian status. Cows were divided in two groups and received 10 microg of buserelin followed by 500 microg of cloprostenol 7 days apart starting from 21 (GnRH21, n=7) or around 37 days postpartum (GnRH37, n=7). The groups were further classified according to presence (-CL) or absence (-NCL) of functional corpora lutea (CL) on the day of GnRH treatment (d 0): GnRH21-NCL (n=4), GnRH21-CL (n=3) and GnRH37-CL (n=7). Ovarian morphology was monitored and the concentrations of P(4), E(2), FSH and insulin-like growth factor 1 (IGF-1) were measured. All cows ovulated after administration of GnRH. The P(4) levels of the GnRH21-NCL group from d 0 to d 5 were lower than those of the GnRH21-CL (P<0.05) and GnRH37-CL groups (P<0.01). In contrast, the E(2) levels of the GnRH21-NCL group within d 2 to d 6 were higher (P<0.05) than those of the other groups. Compared with the GnRH37-CL group, the GnRH21-NCL group had more small follicles on d 2 (P<0.05), d 3 (P<0.01) and d 4 (P<0.01) and more large follicles on d 5 (P<0.05). The induced CL and new ovulatory follicles were larger in the GnRH21-NCL group compared with the GnRH21-CL (P<0.001 and P<0.01) and GnRH37-CL groups (P<0.001 and P<0.05). IGF-1 did not differ among the groups. The GnRH21-NCL group had higher FSH levels than the GnRH21-CL (P<0.01) and GnRH37-CL groups (P<0.001) on d 0. Low P(4) and high FSH levels may suggest higher gonadotropin support on the enhanced ovarian morphology of the GnRH21-NCL group. PGF(2 alpha) treatment induced CL regression and subsequent ovulation in 3/4 (75%), 3/3 (100%) and 7/7 (100%) cows in the GnRH21-NCL, GnRH21-CL and GnRH37-CL groups, respectively. In conclusion, a 7-day GnRH-PGF(2 alpha) synchronization protocol can effectively induce dairy cows to ovulate as early as 21 days postpartum, regardless of ovarian status.

Pancytopenia with bleeding tendency associated with bone marrow aplasia in a Holstein calf.

An 11-day-old Holstein calf presented with a high rectal temperature and tachypnea. Treatment with antibiotics and non-steroidal anti-inflammatory drugs did not improve the clinical signs. Bleeding tendency, with several hemorrhage spots on the body surface, appeared five days after admission. Severe pancytopenia was observed in the blood examination. The calf died on the 11th day after admission with severe bleeding from an injection site. Necropsy findings revealed that the pancytopenia had resulted from severe bone marrow aplasia. A congenital disorder was suspected to be the cause of pancytopenia associated with bone marrow aplasia.

Changes in plasma metabolic hormone concentrations during the ovarian cycles of Japanese black and holstein cattle.

The aim of the present study was to investigate the changing profiles of plasma metabolic hormones during the ovarian cycles of beef and dairy cattle. We used 16 non-pregnant, non-lactating Japanese Black beef cattle (6 heifers and 10 cows; parity=2.3 +/- 0.8) and 12 multiparous Holstein dairy cows (parity=3.0 +/- 0.3). Blood samples for hormonal analysis (growth hormone, GH; insulin-like growth factor-I, IGF-1; insulin; and progesterone, P4) were obtained twice weekly for 40 days before artificial insemination for Japanese Black cattle and from 50 to 100 days postpartum for Holstein cows. Luteal phases were considered normal if the P4 concentrations for at least 3 time points over the course of 7 days remained above 1 ng/ml and at least 2 of the time points were above 2 ng/ml. The patterns of the ovarian cycles were classified into two types (normal or abnormal, such as having prolonged luteal phase and cessation of cyclicity) on the basis of the plasma P4 profiles. The plasma concentrations of IGF-1 in both breeds increased transiently during the preovulatory period when the P4 levels were low and decreased to lower levels during the luteal phase when the P4 levels were high. The plasma concentrations of insulin in the 3(rd) week of normal ovarian cycles when the plasma P4 concentration dropped to less than 1 ng/ml were higher than those at other time points in the Japanese Black cattle, but not in the Holstein cows. The plasma concentrations of GH did not change during the ovarian cycle in either breed. In conclusion, the present study indicates that the plasma IGF-1 concentration increases during the follicular phase (low P4 levels) and decreases during the luteal phase (high P4 levels) in non-lactating Japanese Black and lactating Holstein cattle. The results suggest that ovarian steroids, rather than nutrient status, may be related to the cyclic changes in IGF-1 secretion from the liver in cattle.

Atresia ani with diphallus and separate scrota in a calf: a case report.

Atresia ani, a common genetic defect in animals, is often accompanied by urogenital defects in calves. This paper reports a case of atresia ani with diphallus and separate scrota in a calf. The calf was born with atresia ani; surgery (to open the anus) was performed 3 days after birth. No urogenital abnormalities were noticed until 4 months after birth. At that time, two separate scrota (each containing a testis) and a sac-like structure in the middle of two scrota, were visible. The gait was abnormal, with abduction of the hind limbs while walking. Additionally, the hind legs appeared wider than usual at the hip joints. Two weeks later, two peni (diphallia) was observed, each in a separate preputial sheath. The calf had a normal karyotype on cytogenetic examination. Plasma concentrations of testosterone at 5.5, 6, and 7 months of age were 3.5, 1.9, and 1.7 ng/ml, respectively. At necropsy (7 months of age), the prepuce was thick and the glans of the right penis was adhered to the prepuce. The left penis did not have a urethra or retractor penis muscles. The sac-like structure in the middle of the two scrota contained the urinary bladder and a loop of small intestine. The pubic bone had failed to fuse at the pelvic symphysis. In conclusion, this is the first reported case of atresia ani with diphallus, separate scrota, and pubic bone separation in a calf.

Effect of active immunization of pony mares against recombinant porcine inhibin alpha subunit on ovarian follicular development and plasma steroids and gonadotropins.

Two pony mares were immunized against recombinant porcine inhibin alpha subunit three times with 39 day intervals. Clinical findings and endocrinological changes before immunization were taken as the control. The first significant rise in the anti-inhibin titre (P<0.05) in the circulation was found 27 days after the first injection. Maximum binding activity was reached by the 12th day after the second booster dose. The number of small, medium and large sized follicles had increased significantly compared to before immunization (11.75 +/- 4.30, 2.75 +/- 0.69 and 2.51 +/- 0.63 vs 6.50 +/- 1.43, 1.83 +/- 0.44 and 1.33 +/- 0.38, respectively), but the ovulation rate remained unchanged after immunization. The average plasma concentration of FSH and estradiol-17beta during the estrous cycle increased significantly (P<0.05) after immunization. These results suggest that immunization against inhibin is a useful tool to increase the number of ovarian follicles during the estrous cycle of pony mares. Moreover, the present study supported the concept that inhibin plays a major role in the control of follicular growth through its inhibitory effect on FSH secretion synergistically with steroid hormones.

Partial purification of mare early pregnancy factor.

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the serum in early pregnancy. We have already reported the development of the rosette inhibition test for mare EPF and have detected EPF in thoroughbreds and ponies. Here, we attempted to purify equine EPF from pregnant mare serum. METHODS OF STUDY: Mare EPF was purified by ultrafiltration and ion-exchange chromatography. Purified EPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and a neutralization test. EPF activity was estimated as the rosette inhibition titer (RIT) by the rosette inhibition test. RESULTS: Purified EPF bound to carboxymethyl (CM) sepharose and did not adsorb to diethylaminoethyl (DEAE) sepharose. SDS-PAGE revealed that in the final purified fraction there were many proteins. In the immunoblotting analysis, a protein band of 25.8 kDa was detected as the pregnancy-specific band. Further, antibody gained from the 20 to 30 kDa protein band of the final purified fraction neutralized the RIT activity of pregnant mare serum. CONCLUSIONS: Mare EPF was detected in the final purified fraction and had a molecular weight of 25.8 kDa. EPF in the mare is similar to that obtained from the serum of pregnant cows.

Endocrinological changes before and after removal of the granulosa theca cell tumor (GTCT) affected ovary in 6 mares.

To clarify the endocrinological characteristics of the mares with granulosa theca cell tumor (GTCT), peripheral plasma samples from the 6 mares affected with GTCT were collected before and after the surgical removal of the affected ovary. Concentrations of testosterone (T), follicle stimulating hormone (FSH), luteinizing hormone (LH), immunoreactive-inhibin (ir-INH), progesterone (P) and estradiol-17beta (E(2)) in the plasma samples were measured by radioimmunoassay. Before removal of GTCT in all cases, the concentrations of T were significantly higher than those of normal mares at the breeding and non-breeding seasons, whereas plasma concentrations of FSH, LH, ir-INH, P and E(2) were lower. After surgical removal of the affected ovary, the circulatory concentrations of T was declined, but the concentrations of other hormones were constantly low as compared with those of normal mares. The present study suggests that 1) the source of higher T may be due to the abnormal follicles in ovary of GTCT, 2) in the case of GTCT the elevated level of T is observed due to the lack of aromatase, and 3) the high level of T is a typical characteristics for GTCT in mares. It is also suggested 4) due to the elevated levels of T the concentrations of gonadotropins may be suppressed.

Localization of inhibin alpha-, betaA- and betaB-subunits and aromatase in ovarian follicles with granulosa theca cell tumor (GTCT) in 6 mares.

To clarify the morphological and immunohistochemical characteristics in mares with granulosa theca cell tumor (GTCT), the localization of inhibin subunits (alpha, betaA, betaB) and aromatase in the granulosa cell layers and theca layers in the ovarian follicles were determined by immunohistochemical staining. The follicles were obtained from the ovaries of 6 mares with GTCT and 4 normal mares as controls. Immunohistochemically, inhibin alpha-subunit was localized in the granulosa cells of all follicles showing different sizes in all GTCT cases and betaA- subunit was localized in two GTCT cases in all sized follicles. But inhibin betaB- subunit and aromatase were not localized in GTCT cases. On the other hand, inhibin alpha-, betaA-, and betaB-subunits and aromatase were localized in the large and medium sized follicles, but inhibin betaA- and betaB-subunits and aromatase were not stained in the small sized follicles in normal cases. These findings suggest that some mares with GTCT can secrete dimeric inhibin (inhibin A), but all GTCT cases cannot secrete inhibin B. By the results of aromatase staining it is clear that testosterone is not converted into estradiol due to the lack of aromatase in the GTCT follicles.

Spontaneous repair of the atrophic contralateral ovary without ovariectomy in the case of a granulosa theca cell tumor (GTCT) affected mare.

A 21 year old thoroughbred mare with granulosa theca cell tumor (GTCT) in the right side and atrophic contralateral ovary was investigated in this study. After arrival at our laboratory on 10th December 1999, the clinical diagnosis of GTCT was examined by rectal palpation and ultrasonographic image of ovaries. Plasma from peripheral blood was collected in the breeding and non-breeding seasons for hormonal analysis. The results showed that the contralateral ovary regained normal activity without any treatment of the GTCT affected ovary and contained follicles showing different sizes 19 months later. However, the affected right ovary, which became smaller after 4 months, was totally inactive without any follicle. The observations clearly demonstrate that without any treatment of the GTCT affected ovary, a mare can return to her normal estrous cycle within a certain period in some GTCT cases.

Application of timed artificial insemination protocols to grazing Japanese black cattle with long open period.

To investigate the viability of timed artificial insemination (TAI) protocols in grazing Japanese Black cattle with long open period, ovarian status and progesterone and estradiol-17 beta profiles of the animals during the protocol were monitored. In 1998, prosta-glandin F(2a) (PG) was administered to 36 animals seven days after GnRH injection. Three out of the 36 animals were inseminated after detection of estrus and did not receive further treatment. The second GnRH was injected to the remaining 33 animals 48 hr after the PG injection and TAI was performed 24 hr later. In 1999, PG was injected to 25 animals six days after GnRH, the second GnRH was injected to 22 animals 48 hr after PG, and TAI was performed 16 hr later (The other three animals were inseminated before the time of TAI). The percentage of the animals with at least one functional corpus luteum and one follicle equal to or greater than 10 mm in diameter at PG injection was similar between the groups in 1998 and 1999. Likewise, the hormonal profiles were similar between the two groups. Pregnancy rates (PR) after the TAI protocols and natural mating in 1998 and 1999 were 75.0% and 88.0%, respectively. These figures were comparable to the PR obtained by conventional estrus synchronization protocols using PG (in 1995; 69.4%) or CIDR (in 1996; 59.1%). In conclusion, the TAI protocol can be applicable into grazing Japanese Black cattle with long open period.

Comparison of the effectiveness of ovulation synchronization protocol in anestrous and cycling beef cows.

Applicability of ovulation synchronization protocol using GnRH and PGF(2alpha) (PGF) injection to anestrous beef cows remains controversial. We compared the effectiveness of the protocol in the anestrous stage of the beef cow with that in the cycling stage using the same animals. Ovaries of five Japanese Black and three Japanese Shorthorn cows were ultrasonographically examined, and blood samples were collected daily for hormonal analyses. Each animal received the protocol twice (Day -6 to -8: GnRH, Day 0: PGF, Day 2: GnRH). Additional blood samples were taken before and after GnRH injection for LH and FSH measurements to evaluate the pituitary function. For the ovarian status at the onset of the protocol cows were divided into anestrous (n=8) and cycling (n=8) stages. There was no significant difference in size of the dominant follicle at the first and second GnRH injections, and in the magnitude of the pituitary response to GnRH between the two stages. However, the size of the corpus luteum and progesterone concentrations at the PGF injection in the anestrous stage were significantly smaller and lower (P<0.01), respectively, and ovulation synchronization rate in the anestrous stage was significantly lower (P<0.05) than in the cycling stage. In conclusion, ovulation synchronization protocol in anestrous beef cows has limited effectiveness.

Origin of plasma insulin-like growth factor-I (IGF-I) during estrus in goats.

The objective of this study was to clarify the origin of the increase in plasma insulin-like growth factor-I (IGF-I) during estrus in goats. Focusing on the uterus, the effect of estradiol-17 beta (E2) on the secretion of IGF-I was examined using ovariectomized and hysterectomized animals. A single 5 microg/kg BW of E2 was injected intramuscularly into ovariectomized and hysterectomized goats for 3 consecutive days, and plasma IGF-I concentrations in the two groups were compared. The concentrations of IGF-I rose after the treatments in both groups. The concentrations were significantly higher from 3 to 8 days after the treatment than before the treatment in ovariectomized goats (P<0.05), and from 1 to 3 days after the treatment than before in hysterectomized goats (P<0.05). Thus higher concentrations of plasma IGF-I tended to last longer in ovariectomized than hysterectomized goats. The area under the IGF-I response curve for the 8-day period after the first injection of E2 tended to be greater in ovariectomized than in hysterectomized goats. The results show that E2 increases plasma IGF-I concentrations in goats, and suggest that E2-stimulated IGF-I in plasma may originate mainly from the uterus.