Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

Pseudorabies virus infection in hunting dogs in Oita,Japan: Report from a prefecture free from Aujeszky’s disease in domestic pigs

We isolated two pseudorabies virus (PRV) isolates (designated OT-1 and OT-2) from two hunting dogs exhibiting neurological manifestations after eating the flesh of wild boar hunted in Oita prefecture, Kyushu Island, Japan. The isolates corresponded to a previously reported PRV (MY-1 strain) isolated from a hunting dog in neighboring Miyazaki prefecture, and it clustered into genotype II based on the glycoprotein C sequence. Our results suggest that this common PRV strain may have been maintained in wild boars on Kyushu Island even though domestic pigs in this area have attained an Aujeszky’s disease-free status.     

The W‐ and Z‐linked EE0.6 sequences used for molecular sexing of captive Japanese crested ibis on Sado Island

The Japanese crested ibis Nipponia nippon is a critically threatened bird. Accurate sexing is necessary to perform effective management of captive breeding toward a national project for a tentative release of the Japanese crested ibis on Sado Island. A PCR‐based sexing method targeting a 0.6 kb EcoRI fragment (EE0.6) sequence on W chromosome with AWS03 and USP3 primers has been developed for the Japanese crested ibis. However, the primers were selected from the EE0.6 sequences from bird species other than the Japanese crested ibis. In this study, we determined the W‐ and Z‐linked EE0.6 sequences in the Japanese crested ibis, and clarified Japanese crested ibis sequence mismatch in the binding sites of the primers. Further, we found no polymorphism in the primer binding sites among five founder birds for the Sado captive Japanese crested ibis population. These findings validated the PCR‐based sexing method with the AWS03 and USP3 as accurate molecular sexing methods of captive Japanese crested ibis on the Sado Island. Additionally, we designed a primer set for a novel PCR‐based sexing, based on the EE0.6 sequences obtained in this study. This novel sexing method may be useful for future ecological research following the release of Japanese crested ibis on Sado Island. This is the first report to show the EE0.6 sequences in Japanese crested ibis.     

Nutritional factors that regulate ovulation of the dominant follicle during the first follicular wave postpartum in high-producing dairy cows

During recent decades, milk production per cow has increased drastically due to improved management, nutrition, and genetic selection; however, the reproductive performance of high-producing dairy cows has been declining. One of the factors responsible for this low reproductive performance is negative energy balance (NEB). NEB affects the onset of first ovulation in early postpartum cows. It is generally accepted that early first ovulation positively relates to the resumption of normal ovarian function, first service, and conception rate in dairy cows. Hence, delayed first ovulation has a negative impact on subsequent fertility. The metabolic condition of cows in NEB shifts to catabolic metabolism, which in turn causes increased plasma growth hormone and non-esterified fatty acid concentrations and decreased plasma insulin-like growth factor-1, insulin, and glucose concentrations. On the other hand, plasma β-carotene concentrations decrease throughout the dry period and reach their nadir in about the first week postpartum, and this change reflects energy balance during the peripartum period. β-Carotene plays a role independently of vitamin A in the reproductive performance of dairy cows, and the positive relationship between supplemental β-carotene and reproductive function has been demonstrated in many studies during the past decades. However, β-carotene content in corn silage, which is a popular main feed in high-producing dairy cows, is very low. This review describes nutritional factors related to ovulation during the first follicular wave postpartum in dairy cows.     

Relationship between the first ovulation within three weeks postpartum and subsequent ovarian cycles and fertility in high producing dairy cows

The aim of this study was to investigate the relationship between the first ovulation within 3 weeks postpartum and subsequent ovarian cycles and fertility in high producing dairy cattle in Hokkaido, Japan. In Experiment 1, 110 cows (44 primiparous and 66 multiparous) were used to determine the effects of the first ovulation within 3 weeks postpartum on subsequent ovarian cycles. Milk samples were collected twice weekly from 7 to 100 days postpartum. The first ovulation was identified by an increase in milk progesterone (P4) to more than 1 ng/ml within 3 weeks postpartum. The numbers of cows showing ovulation and anovulation within 3 weeks postpartum were 31 (70.5%) and 13 (29.5%) in the primiparous cows and 35 (53.0%) and 31 (47.0%) in the multiparous cows, respectively. The patterns of ovarian resumption after calving were classified into two types (normal ovarian cycles and abnormal ovarian cycles) on the basis of milk P4 concentrations. Initiation of normal ovarian function in cows ovulated within 3 weeks postpartum occurred earlier than in anovulated cows regardless of the number of calvings (primiparous, 27.8 days vs. 44.4 days; multiparous, 30.6 days vs. 55.7 days; P<0.01). Out of the multiparous cows that ovulated within 3 weeks postpartum, initiation of normal ovarian function followed by a normal luteal phase was earlier than when it was followed by an abnormal luteal phase (25.5 days vs. 40.4 days; P<0.05). Milk P4 concentrations after the first ovulation were lower than those after the second ovulation in both the primiparous and multiparous cows (P<0.05). In Experiment 2, 22 multiparous cows were used to determine the effects of the first ovulation within 3 weeks postpartum on subsequent fertility. Blood samples were collected once a week from 0 to 3 weeks postpartum. The interval from parturition to first service in ovulated cows was shorter than in anovulated cows (68.4 days vs. 94.8 days; P<0.05). The conception rate by 100 days after calving tended to be higher in ovulated cows than in anovulated cows (50.0% vs. 16.7%, P=0.09). In conclusion, our data strongly suggests that ovulation within 3 weeks postpartum is a crucial phenomenon for subsequent resumption of ovarian function and conception, and thus it can be used as an index of subsequent reproductive performance.     

Application of time-resolved fluorometry to immunoassays for bovine reproductive hormones

The principle of time‐resolved fluorometry with lanthanide chelates was established in the 1980s, but in the field of animal sciences it has not been widely applied to immunoassays. However, immunoassays that utilize time‐resolved fluorometry are possible alternatives to radioimmunoassays, since they can attain high sensitivity without safety risks. In this short review, we introduce the development of time‐resolved immunoassays for inhibin A, inhibin B and follicle‐stimulating hormone (FSH), and describe their application to the investigation of FSH regulation in male and female cattle. The results obtained using these newly developed immunoassays indicate that inhibin A acts as a feedback regulator for FSH secretion in female cattle, whereas inhibin A, and probably inhibin B, do so in male cattle.     

Comparison of estrus induction and subsequent fertility with two different intravaginal devices in ewes during the non-breeding season

Two experiments were conducted to compare the effect of estrus induction by controlled internal drug release (CIDR) and intravaginal cream containing 500 mg progesterone (P cream) in ewes during the non-breeding season. In the first experiment, twenty-four ewes were randomly grouped for two treatments with the different intravaginal devices for 12 days: Group A was the CIDR group and Group B was the P cream group. Blood was collected from all treated ewes, and progesterone (P(4)), estradiol 17-beta (E(2)) and luteinizing hormone (LH) concentrations were measured by enzyme immunoassay. In the second experiment, the conception rates from natural mating, estrus-detected AI (inseminated 12 h after estrus detection), or fixed-time AI (inseminated 42 h after removal of an intravaginal device) in 127 ewes treated with CIDR or P cream were compared. In Experiment 1, the rate of estrus induction and the time of estrus onset after device removal were 91.7% and 36.3 +/- 15.7 h in Group A, and 100% and 35.0 +/- 12.6 h in Group B, respectively. There were no significant differences between the devices. The mean plasma P(4) concentration in Group B was significantly (P < 0.01) lower than Group A between day -9 and day -1 (Day 0: the day of device removal). However, no significant differences were found in the mean E(2) concentrations of the two groups after treatment. The mean time of estrus onset in ewes with an observed LH surge and the time of LH surge after treatment were 23.3 +/- 8.7 h and 30.3 +/- 5.0 h for Group A and 27.6 +/- 6.5 and 26.3 +/- 8.0 h for Group B, respectively, and there were no significant differences. However, a significant difference (P < 0.05) was found in the mean time from the time of estrus onset to LH surge between Group A (6.4 +/- 6.7 h) and Group B (-1.3 +/- 4.1 h). In Experiment 2, the conception rates for natural mating, estrus-detected AI, and fixed-time AI were 55.0, 29.4, and 25.0% for Group A and 40.7, 25.0, and 42.1% for Group B, respectively, and there were no significant differences. These results suggest that the effect of induction of estrus and ovulation and the rate of conception after treatment were comparable to CIDR even though the plasma P(4) concentration of the P cream method tended to be low during the insertion period.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.