The effectiveness of different hormone protocols for improving ovarian function before ovum pick-up in crossbred Japanese black cattle.

Background and Aim: Producing and transferring embryos in vitro are profitable for enhancing premium beef genetics. Reducing costs and enhancing the effectiveness of hormone protocols before ovum pick-up (OPU) yield advantages. This study aimed to confirm that estradiol benzoate (EB) treatment resulted in more medium- and large-sized follicles before OPU and of higher oocyte quality after OPU than non-hormonal treatments, comparable to those undergoing gonadotropin-releasing hormone (GnRH) with follicle-stimulating hormone (FSH) plus prostaglandin F2α (PGF2α) and progesterone-releasing controlled internal drug release (CIDR) or EB with progesterone-releasing CIDR hormonal treatments. Materials and Methods: 30 crossbred Japanese black cows were divided into five equal groups, which were either untreated or treated with different hormone protocols before OPU. Group 1 (cows in estrus) and group 2 (cows in diestrus) were the untreated controls. Cows in group 3 were treated with GnRH + FSH + CIDR + PGF2α. Cows in group 4 received EB, and those in group 5 received EB + CIDR + PGF2α. After administering the protocols, all cow follicles were examined through ultrasonography and categorized by size. Subsequently, all cows underwent OPU, and the oocytes were collected and graded from A to D according to standard criteria. Results: Group 3 presented the highest large follicle numbers, and groups 3-5 had more medium follicle numbers, not different among groups but they had this parameter more than those of control groups 1 and 2. Moreover, groups 3-5 did not differ in combined grades A + B oocytes (good-quality oocytes). Groups 3 and 4 had more A + B oocytes than control groups 1 and 2, whereas group 5 was not different in this parameter from group 1. Conclusion: Among the three hormone protocols, EB treatment proved the most cost-effective and efficient, yielding more high-quality oocytes compared to the non-treatment protocols. To reduce the limitations of EB use in the future, this study suggests researching natural EB phytoestrogens as alternative treatments for improving ovarian function before OPU in other cattle breeds.

Changes in immunoglobulin A and immunoglobulin G concentrations in the saliva of Japanese Black beef cows during calving: A pilot study.

Immunoglobulin A (IgA) in saliva, mostly consisting of secretory IgA, plays an important role in the mucosal immune mechanism. This study evaluated changes in salivary IgA and Immunoglobulin G (IgG) concentrations in Japanese Black cows (n = 16) during calving. Individual saliva samples were collected -2, 0, and 2 weeks postpartum. Immunoglobulin concentrations differed significantly among weeks (P < 0.05), but the effect of parity and week × parity was insignificant. Salivary IgA concentrations decreased drastically (P < 0.05) after calving compared with those at -2 weeks postpartum and remained low until 2 weeks postpartum. The salivary IgG concentrations decreased gradually during peripartum and differed at -2 and 2 weeks postpartum (P < 0.05). Considering the immunoglobulin concentrations at -2 weeks postpartum as the reference standard for 100%, the rates of decrease in IgA concentrations (36.7 ± 6.9%) were significantly lower (P < 0.05) than those of IgG (70.3 ± 10.1%) at calving day. To our knowledge, this is the first report indicating that salivary IgA concentrations decreased drastically after calving in Japanese Black cows. Further studies monitoring the secretory functions of IgA in the salivary gland are essential for understanding maternal immunity in cattle.

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan, and analysis of the metabolic profiling and reproductive performance of heterozygous cows.

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.

Establishment of long-term chronic recording technique of in vivo ovarian parenchymal temperature in Japanese Black cows.

The reproductive performance of cattle can be suppressed by heat stress. Reproductive organ temperature, especially ovarian temperature, may affect follicle development and ovulation. The establishment of a technique for long-term measurement of ovarian temperature could prove useful in understanding the mechanisms underlying the temperature-dependent changes in follicular development and subsequent ovulation in cows. Here we report a novel method facilitating long-term and continuous recording of ovarian parenchymal temperature in cows. The method revealed that the ovarian temperature in the luteal phase was constantly maintained lower than the vaginal temperature, and that the diurnal temperature variation in the ovary was significantly greater than that in the vagina, suggesting that the ovaries may require a lower temperature than other organs to maintain their functions. This novel method could be used for the further understanding of ovarian functions during estrous cycles in cows.

Effect of post artificial insemination treatment with two different progesterone intravaginal devices on conception and synchronization of the returning estrus in Japanese Black cows.

The objective of this study was to evaluate the effects of post artificial insemination (AI) treatment with intravaginal progesterone device (P4 device) on conception rate, synchronization of returning estrus and plasma P4 concentration in Japanese Black cows. Nineteen cows were treated with DIB (1.0 g P4) from Day 12 to 19 (Day 0=day of the first AI), 27 cows were treated with a CIDR (1.9 g P4) from Day 12 to 19, and 33 cows were not treated after the first AI (control). Estrous behavior was daily examined between Day 20 and 25, and cows returning to estrus were inseminated (the second AI). On Day 19, plasma P4 concentration was not different among DIB, CIDR and control groups. There was no significant difference in conception rate after the first AI among three groups (DIB: 63.2%, CIDR: 66.7% and control: 72.7%). In non-pregnant cows, there was no significant difference in the proportion of cows showed returning estrus between Day 20 and 25 (DIB: 57.1%, CIDR: 22.2% and control: 44.4%), and day of returning estrus was not synchronized. The overall conception rate after the first and second AI was not different among the groups. In conclusion, post-AI treatment with intravaginal devices containing 1.0 and 1.9 g P4 from Day 12 to 19 neither increased plasma P4 concentration nor improved fertility and synchronization of the returning estrus in Japanese Black cows.

Effects of supplemental ß-carotene on colostral immunoglobulin and plasma ß-carotene and immunoglobulin in Japanese Black cows.

Data from 26 Japanese Black cows were collected to clarify the effects of supplemental ß-carotene on colostral immunoglobulin (Ig) and plasma ß-carotene and Ig in the cows. Cows were assigned to control or ß-carotene groups from 21 days before the expected calving date to 60 days after parturition. Supplemental ß-carotene was provided at 500 mg/day in the ß-carotene group. Supplemental ß-carotene drastically increased plasma ß-carotene concentrations in the cows from parturition to 60 days after parturition, and plasma ß-carotene concentrations in the control and ß-carotene groups at parturition were 202 and 452 µg/dl, respectively. Supplemental ß-carotene had no effects on plasma IgG1 , IgA or IgM concentrations at parturition. Supplemental ß-carotene increased colostral IgG1 concentrations in the cows, but colostral ß-carotene, IgA and IgM concentrations were not affected by supplemental ß-carotene. These results indicate that supplemental ß-carotene is effective to enhance colostral IgG1 concentrations and plasma ß-carotene concentrations in Japanese Black cows.

Association of interservice interval with conception rate in Japanese Black cattle.

This study aimed to clarify the association of interservice interval (ISI) with conception rate (CR) and to evaluate factors affecting ISI in Japanese Black cattle. Data used in this study covered 32,639 artificial insemination (AI) records on 972 farms. The mean ± SEM of ISI and CR was 57.0 ± 0.3 days and 44.6 ± 0.3%, respectively. The relative frequency of ISI differed significantly between parity groups (p < .05). For parity 0, the proportion of ISI at an 18- to 24-day interval was 43.6%, whereas the proportions in the other parity groups ranged from 27.2% to 29.1%. The CR was associated significantly with ISI, parity and AI number (p < .05), but not with season. Cows reinseminated at an 18- to 24-day interval had the highest CR (46.8%; p < .05). Cows reinseminated at a 39- to 45-day interval had a similar CR to those reinseminated at 25- 38-day and 46- to 59-day intervals. Cows reinseminated at 11-17, 60-66 and 67 days or longer intervals had the lowest CR (p < .05). Thus, cows returning to oestrus in a normal cycle tended to have a higher probability of conceiving compared with those that returned to oestrus after a normal cycle for any degree of parity and at any number of AI.

Digestibility of common reed (Pharagmites communis Trin.) silage as ruminant feed and effects of inclusion levels in the diet of breeding cows on feed intake, ruminal fermentation and blood metabolites.

To examine the digestibility of common reed (Pharagmites communis Trin.) silage and effects of inclusion levels in the diet of breeding cows on nutrient intakes, ruminal fermentation and nutrient status, two digestion trials using four sheep and cows, respectively, at the maintenance stage were conducted. In the trial using sheep, common reed silage contained 20% crude protein (CP) and 50% total digestible nutrients (TDN) on a dry matter basis. In the trial using cows, treatments were the control (92.4% Sudangrass hay and 7.6% soybean meal) and CS25, CS50 and CS80 (replacing 25%, 50%, and 80% of the control with silage, respectively). The intake and sufficiency rate of CP increased by increasing the silage level in the diet (P < 0.05), and the CP requirements of cows at maintenance was satisfied in CS80. In contrast, the sufficiency rate of TDN decreased in CS80, although it was 99.8%. Ruminal pH and NH3 -N concentration changed among the treatments (P < 0.05); however, the blood metabolites were not affected by silage. The results indicated that including common reed silage of up to 80% in the diet of breeding cows was possible and did not have adverse effects on the blood metabolites.

Effects of inclusion levels of pelleted silvergrass (Miscanthus sinensis Andress.) in the diet on digestibility, chewing activity, ruminal fermentation and blood metabolites in breeding Japanese Black cows.

To examine the effects of inclusion levels of pelleted silvergrass (PS) in the diet on digestibility, ruminal fermentation and nutrient status of breeding Japanese Black cows, four cows were allotted to a 4 × 4 Latin square design experiment. Treatments were control fed a diet consisting of 89.4% Sudangrass hay and 10.6% soybean meal on a dry matter (DM) basis, and PS18, PS27 and PS45 fed the diet replaced with 18%, 27% and 45% of control with PS, respectively. The total digestible nutrients (TDN) content of PS was 45.6% on a DM basis. The TDN intakes were significantly decreased by increasing PS level in the diet (P < 0.05), but were higher than the TDN requirement of maintenance cows in all treatments. The total chewing time was decreased significantly by increasing PS level in the diets (P < 0.05). However, the pH and concentration of volatile fatty acid in the ruminal fluid and serum metabolite concentrations were not significantly different among the treatments. The results suggested that including PS up to 45% in the diet did not have adverse effects on the ruminal fermentation and nutrient status in breeding Japanese Black cows at the maintenance stage.

Effect of heat stress on age at first calving of Japanese Black cows in Okinawa.

Calving records from birth certificates of cows were analyzed to investigate the effect of heat stress on age at first calving (AFC) of Japanese Black cows. The data set covered 20 years (1990-2009) of calving records. Total number of records was 9279. Daily weather information from weather stations in the vicinity of the farms was used. Temperature-humidity index (THI) fitted to a linear model covered 30 days pre-insemination to 61 days post-insemination. Statistical analysis was conducted with procedures of SAS/STAT. Preliminary analysis showed that THI of the lowest temperature and humidity was most conducive to AFC. Covariance analysis, including main effect of sire, farm and year of insemination and covariates of THI on days showed that regression coefficients of THI on day -7, day -2 and day +31 were statistically significant. The estimated piecewise regression line showed different responses of AFC to THI on days: roof-shasped downward trend on day -7, hockey-stick shaped upward trend on day -2 and day +31. The difference among the estimated regression lines may be caused by direct and indirect factors on reproduction: indirect effect of reduced feed intake, failure of conception at previous insemination, direct effect of heat stress on oocyte and embryo development.

Effects of nutritional status on hormone concentrations of the somatotropin axis and metabolites in plasma and colostrum of Japanese Black cows.

We aimed to determine the effects of nutritional status on concentrations of somatotropic axis hormones (growth hormone (GH) and insulin-like growth factor 1 (IGF-1)), insulin and metabolites (glucose, total protein and nonesterified fatty acids (NEFA)) in the plasma and colostrum in late antepartum cows. Eight pregnant Japanese Black cows were randomly assigned to two experimental groups (n = 4 per group). Control cows (CON) received 100% of their nutritional requirements until parturition, whereas restricted group cows (RES) received 60% of their nutritional requirements. Blood samples were taken during the antepartum period, and blood and colostrum samples were collected on days 0, 1, and 3 after calving. Compared to the CON group, the RES group had higher concentrations of GH and NEFA in plasma, but significantly lower concentrations of glucose and insulin in plasma. The concentrations of GH in plasma after calving were significantly higher, but total plasma protein was significantly lower in RES than in CON cows. Compared to the CON group, the RES group had significantly higher concentrations of GH in colostrum, but significantly lower total concentrations of protein in colostrum. Concentrations of IGF-1 were not different between the two groups. These findings suggest that maternal nutritional status during late gestation influences concentrations of GH and total protein in the blood and colostrum of Japanese Black cows.

Responsiveness of beef cattle (Bos taurus) to human approach, novelty, social isolation, restraint and trade-offs between feeding and social companionship.

This study investigated responsiveness of beef cattle to various environmental stimuli by subjecting 15-16 Japanese Black cows to five tests repeated twice. Within individual behavioral measures, cows were moderately (repeatability = 0.54-0.70) or highly (repeatability = 0.74-0.89) consistent in flight distance during grazing and resting in the human approach tests, maximum distance from the group pen and the number of total and different feed tub visits in the feeding-sociability trade-off test, and unwillingness to enter the restraint, movement under restraint and flight speed after release from restraint in the social isolation and restraint test. By contrast, cows were not consistent in the latency to make the first contact and the number of contacts with novel object(s) in the novelty test (repeatability = 0.24-0.39). Across behavioral measures in different tests, cows showed no consistency (P ≥ 0.05) in any combinations of measures from the two human approach tests, the trade-off test and the social isolation and restraint test. In conclusion, human approach (particularly during resting), feeding-sociability trade-off and social isolation and restraint situations can be used for evaluating personality in Japanese Black cows, while the value of the novelty test needs to be reexamined.

Relationships between immunoglobulin M and immunoglobulin G or A in colostrum of Japanese black multiparous cows.

This study was conducted to clarify the relationships among immunoglobulin (Ig)M, IgG, IgA, ß-carotene, vitamin A and α-tocopherol contents in colostrum of 24 Japanese Black multiparous cows in order to evaluate the role of IgM on colostral IgG and IgA production. Compared with colostral IgG, colostral IgM and IgA were very low but varied widely. There was positive correlation between colostral IgM and IgG, but colostral IgM was not related with colostral IgA. There was no relationship between colostral IgM and age of cows, although colostral IgG was increased with aging. There were positive correlations among colostral ß-carotene, vitamin A and α-tocopherol and these vitamins were positively correlated with colostral IgM and IgG. These results indicate that fat-soluble vitamins may affect colostral IgG and IgM in cows and colostral IgG increases with the increase of colostral IgM.

Nutrient sufficiency in beef cows grazing on a dwarf bamboo-dominated forestland pasture in Japan.

The nutritional status of cows grazing on a 120-ha public alpine forestland pasture dominated by the dwarf bamboo Sasa senanensis located in central Japan was evaluated over the course of 2 years. Data were collected during grazing seasons in three periods: early (June), mid- (August) and late (late September-early October) periods. During these periods, the number of experimental Japanese Black cows varied between three and six. With the exception of the dry matter (DM) intake in 2005 and the crude protein (CP) digestibility in 2006, the DM and CP intake and digestibility were lower in the mid- and late periods than in the early period for both years (P < 0.05). Metabolizable energy intake was slightly insufficient for the requirement of the cows in the late period of 2005; in the mid- and late periods of 2006, the metabolizable energy intake was 0.4 to 0.5 times the requirement. These results suggest that the energy intake of cows grazing on forestland pasture dominated by S. senanensis would not satisfy their requirements starting in the mid-period (August) grazing, even though the pasture had abundant herbage resources during these periods.

Superovulatory response in Japanese Black cows receiving a single subcutaneous porcine follicle-stimulating hormone treatment or six intramuscular treatments over three days.

To reduce labor for superovulation treatment by twice-daily intramuscular (im) administration of FSH for more than 3 to 4 days, we investigated the superovulatory responses of Japanese Black cows to porcine FSH (pFSH) used as a single subcutaneous (sc) administration at two different doses in two different volumes of saline. In experiment 1, 20 Armour units (AU) of pFSH dissolved in either 10 mL (treatment A; n = 14) or 50 mL (treatment B; n = 14) of saline was administered subcutaneously in the neck region. In experiment 2, 30 AU of pFSH dissolved in either 10 mL (treatment C; n = 15) or 50 mL (treatment D; n = 15) of saline was administered subcutaneously in the neck region. The control animals in experiment 1 (n = 14) and experiment 2 (n = 15) received 20 AU of pFSH administered intramuscularly twice daily in decreasing doses for more than 3 days. In experiment 1, mean (±SEM) numbers of CL (15.4 ± 2.5, 18.1 ± 3.4, and 17.2 ± 2.6), total number of ova and embryos (12.9 ± 1.4, 15.9 ± 3.5, and 16.2 ± 2.8), and transferable embryos (7.5 ± 2.0, 10.4 ± 2.8, and 8.0 ± 2.1) did not differ among treatments A, B, and control. In experiment 2, mean (±SEM) numbers of CL (20.5 ± 4.3, 20.4 ± 2.7, and 20.1 ± 3.4), total number of ova and embryos (21.7 ± 4.2, 17.3 ± 3.4, and 16.5 ± 3.2), and transferable embryos (8.1 ± 1.6, 9.3 ± 2.2, and 9.5 ± 1.9) did not differ among treatments C, D, and control. Although there were no differences in serum pFSH concentrations among the three treatments at each of the time points in experiment 1, in experiment 2, the serum pFSH concentration at 6 and 8 hours after pFSH administration in treatment C (3.1 ± 0.8, 2.7 ± 0.5 ng/mL, mean ± SEM) was significantly greater (P < 0.05) than in the control (0.7 ± 0.1, 1.1 ± 0.2 ng/mL). At 10 hours after administration, the pFSH concentration had decreased and there were no differences among the three treatments at subsequent time points. These results suggest that increasing the volume of saline or the dose of pFSH does not affect the absorption pattern of pFSH administered as a single sc administration. In conclusions, single sc administration of pFSH at a dose of 20 or 30 AU dissolved in 10 or 50 mL of saline is able to induce a superovulatory response comparable with that obtained by twice-daily im administration in Japanese Black cows.

Relationships between immunoglobulin and fat-soluble vitamins in colostrum of Japanese Black multiparous cows.

Data from 19 Japanese Black multiparous cows were collected to clarify the relationships among immunoglobulin (Ig) G, IgA, ß-carotene, vitamin A and α-tocopherol contents in colostrum of cows in order to evaluate the role of fat-soluble vitamins on colostral IgG and IgA production. Mean colostral IgG was 141 mg/mL, ranging from 65 to 208 mg/mL, whereas mean colostral IgA was 8.7 mg/mL, ranging from 1.0 to 34.6 mg/mL. Colostral IgG increased with aging in multiparous cows. There were positive correlations between colostral IgG and colostral vitamin A or colostral α-tocopherol in cows, and the higher adjusted R(2) was obtained in the prediction model of colostral IgG from age and colostral vitamin A. Colostral vitamin A was positively correlated with colostral ß-carotene or colostral α-tocopherol in cows, but there were no relationships between colostral IgA and colostral IgG or colostral fat-soluble vitamins. These results indicate that fat-soluble vitamin contents in colostrum of cows may change in similar patterns and high colostral vitamin A is related with high colostral IgG.

Increased ectopic fat cells in the longitudinal muscularis layer of the oviduct isthmus in obese Japanese Black cows.

In obese humans, mesenchymal stem cells differentiate to become ectopic fat cells in muscles. These ectopic fat cells inhibit the contraction of vascular smooth muscles. Stem cells have been recently identified in the human oviduct, a structure important in reproduction. We therefore investigated the number of Oil Red O (ORO)-positive cells in the oviducts of control Japanese Black cows (n = 6; body condition score [BCS], 3.0 on a 5-point scale) compared to those with diet-induced obesity (n = 5; BCS, 4.0). We stained the ampulla and isthmus collected on the second day after ovulation with ORO and then counted the positive cells in each layer in 10 cross-sections of the ampulla or isthmus. The obese group (23.4 ± 3.4 in the 10 sections) had larger numbers of ORO-positive cells in the longitudinal muscularis of the isthmus (P < 0.05) than did the control group (15.0 ± 2.4). ORO-positive cells were also observed in all other layers of the isthmus and ampulla; however, the number of cells in these layers did not differ significantly between obese cows and controls. Whether this observed increase in ORO-positive cells in the oviducts of obese cows affects their reproduction warrants further study.