Development of the Timed Re-Insemination (TRI-synch) program re-inseminating 24 days after the initial service in dairy cows.

For the timed re-insemination at the minimal interbreeding interval, cows were treated with a progesterone (P4 )-releasing intravaginal device from Days 13-15 to 21 post-insemination (Day 0 = estrus), followed by plasma P4 assay on Day 23 and then subjected to the Experiments 1 and 2. In Experiment 1, of 18 cows, 6 cows were determined as luteolysis with low (<1 ng/ml) plasma P4 concentrations on Day 23 and ovulated on Days 24 (3 cows), 25 (1 cow), and 26 (1 cow) except a cow affected by ovarian quiescence. In Experiment 2, all cows were treated with GnRH on Day 23. Cows with low (<1 ng/ml) plasma P4 concentrations on Day 23 were diagnosed as non-pregnant and subjected to the re-insemination in the morning of Day 24 irrespective of estrous signs. Of 36 cows, 15 cows were diagnosed as being non-pregnant on Day 23. Fourteen cows of the non-pregnant animals were re-inseminated in the morning of Day 24 irrespective of estrous signs and the pregnancy rate of re-insemination was 36%. The conception rates of initial and re-inseminations were 50% (18/36) and 36% (5/14), respectively. The overall pregnancy rate by adding the rates of initial and re-inseminations was 64% (23/36).

Different prostaglandin F2 α secretion in response to oxytocin injection between pregnant and non-pregnant cows: effect of the day of oxytocin challenge test for determining the difference.

The present study was conducted to determine the difference in plasma prostaglandin F2 α metabolite concentrations following oxytocin (OT) challenge between pregnant and non-pregnant cows. Experiment 1: cows were subjected to the OT challenge test on days 12, 14 or 16 (day of estrus = day 0) with or without prior insemination and plasma 13,14-dihydro-15-keto prostaglandin F2 α (PGFM) concentrations were measured from -30 to 180 min after OT injection. On day 16, the increment of plasma PGFM concentrations in response to OT injection was significantly smaller in pregnant than that in cyclic cows. On days 12 and 14, there was little OT-induced PGFM secretion and no difference in PGFM increase between the pregnant and cyclic cows. Experiment 2: cows were inseminated on day 0 and subjected to the OT challenge test on day 16. Cows were classified into non-pregnant/early embryonic death (NP/EED), late embryonic death (LED) and pregnant (PREG) groups. The increment of PGFM concentrations in response to OT injection was less in both PREG and LED groups than that in the NP/EED group. In conclusion, plasma PGFM secretion induced by OT is suggested as the base of pregnancy diagnosis prior to returning estrus in cows.

A predictive threshold value for the diagnosis of early pregnancy in cows using interferon-stimulated genes in granulocytes.

Interferon tau plays an important role in establishing bovine pregnancy. Interferon-stimulated genes (ISGs) have been examined to identify a suitable indicator for the diagnosis of early gestation in cows. Although ISGs can be specifically detected in peripheral white blood cells during early gestation, its reliability remains to be validated. In the current study, a predictive threshold level of ISGs to determine pregnancy in cows during Days 20-22 of gestation was verified by analyzing the expression of ISGs in granulocytes and peripheral blood leucocytes (a total of 57 cows were used, 28 of which were pregnant and 29 were non-pregnant). Four genes, interferon-stimulated gene 15 ubiquitin-like modifier (ISG15), MX dynamin like GTPase (MX) 1, MX2, and 2'-5'-oligoadenylate synthetase 1 (OAS1), were analyzed via quantitative RT-PCR and a receiver operating characteristic (ROC) curve was produced to visualize diagnostic accuracy measures. The expression values of the four ISGs during the estrous cycle (100 collection points from 65 cattle) were used to determine a pregnancy prediction cutoff value. Pregnancy status was determined using these cutoff values and then confirmed by ultrasonography. ROC analysis was then applied to confirm the accuracy of the pregnancy statuses (positive and negative) statistically. The statistical evaluation of the diagnostic accuracy measurements suggested that the average values of ISG15 and MX2 in granulocytes were reliable indicators of pregnancy within the three weeks after insemination with 80% accuracy. Average ISG15 and MX2 levels during the estrous cycle were more reliable biomarkers for the prediction of gestation. They predicted negative and positive pregnancies efficiently within three weeks after artificial insemination.

Administration of gonadotropin-releasing hormone agonist on Day 5 increases luteal blood flow and improves pregnancy prediction accuracy on Day 14 in recipient Holstein cows.

This study assessed the effects of gonadotropin-releasing hormone (GnRH) treatment on Day 5 (Day 0 = estrus) on luteal blood flow and accuracy of pregnancy prediction in recipient cows. On Day 5, 120 lactating Holstein cows were randomly assigned to a control group (n = 63) or GnRH group treated with 100 µg of GnRH agonist (n = 57). On Days 3, 5, 7, and 14, each cow underwent ultrasound examination to measure the blood flow area (BFA) and time-averaged maximum velocity (TAMV) at the spiral arteries at the base of the corpus luteum using color Doppler ultrasonography. Cows with a corpus luteum diameter ≥ 20 mm (n = 120) received embryo transfers on Day 7. The BFA values in the GnRH group were significantly higher than those in the control group on Days 7 and 14. TAMV did not differ between these groups. According to receiver operating characteristic analyses to predict pregnancy, a BFA cutoff of 0.52 cm2 yielded the highest sensitivity (83.3%) and specificity (90.5%) on Day 7, and BFA and TAMV values of 0.94 cm2 and 44.93 cm/s, respectively, yielded the highest sensitivity (97.1%) and specificity (100%) on Day 14 in the GnRH group. The areas under the curve for the paired BFA and TAMV in the GnRH group were 0.058 higher than those in the control group (0.996 and 0.938, respectively; P < 0.05). In conclusion, GnRH treatment on Day 5 increased the luteal BFA in recipient cows on Days 7 and 14, and improved the accuracy of pregnancy prediction on Day 14.

Correlation of blood metabolite concentrations and body condition scores with persistent postpartum uterine bacterial infection in dairy cows.

To analyze the relationship of blood metabolite concentrations and body condition score (BCS) with persistent bacterial uterine infection, specifically that caused by Trueperella pyogenes and anaerobic bacteria, uterine bacteriological swabs (n = 128) were collected from 64 Holstein cows at 5 (W5) and 7 (W7) weeks postpartum, and the percentage of neutrophils in the endometrium was evaluated. Blood glucose, total cholesterol (T-cho), blood urea nitrogen (BUN), non-esterified fatty acid (NEFA), and ß-hydroxybutyric acid concentrations were analyzed at 3 weeks (W-3) and 1 week (W-1) prepartum and W3, W5, and W7 postpartum. BCS were evaluated at W-3, W3, and W7. Blood glucose concentrations at W-3 and W-1 in cows with persistent bacterial infection were lower (P = 0.05) than in the rest of the cows. Total BUN concentrations in cows with persistent bacterial infection were lower (P < 0.01) than those in other cows, although the association between the pre or postpartum time and status of infection was not significant. Total NEFA concentrations in cows with persistent bacterial infection were similar to those in uninfected cows and cows positive for infection at W5 but not W7. Total BCS in cows with persistent bacterial infection were lower (P < 0.01) than those in cows positive for infection at both W5 but not W7 and W7 but not W5; however, the association between the pre or postpartum time and status of infection was not significant. Glucose concentrations at W-3 and W-1 negatively correlated with persistent bacterial infection at W5 and W7 (P < 0.01). BUN concentrations at W3 (P < 0.01), W5 (P < 0.05), and W7 (P < 0.05) and BCS at W3 (P < 0.01) negatively correlated with persistent postpartum bacterial infection. Decreased prepartum blood glucose concentrations might be an important risk factor for persistent postpartum bacterial uterine infection in dairy cows.

Pregnancy prediction on the day of embryo transfer (Day 7) and Day 14 by measuring luteal blood flow in dairy cows.

This study aimed to assess the suitability of luteal blood flow analyses measured by color Doppler ultrasonography (CDUS), to predict pregnancy at pre- and post-embryo transfer (ET) in dairy cows, and to compare with the established criterion like luteal size and plasma progesterone (P4) concentrations. Lactating Holstein cows (n = 65) with spontaneous (n = 34) or synchronized estrus (n = 31) were examined. Cows with a CL greater than or equal to 20 mm in diameter (n = 58) received embryo transfer on Day 7 (Day 0 = estrus). Brightness mode images were captured for calculation of the CL area, luteal cavity area, and dominant follicle area on Days 3, 5, 7, and 14. Color Doppler ultrasonography examinations were conducted to determine the blood flow area (BFA) within the CL at the maximum diameter and the time-averaged maximum velocity (TAMV) of the base of the spiral artery on the same days. Plasma P4 concentrations were determined from blood samples collected at each ultrasound examination. Pregnancy was diagnosed by an ultrasound on Day 30. There was no significant difference in the proportion of cows received embryo (91.2% vs. 87.1%, P = 0.70) and pregnancy rate (58.1% vs. 59.3%, P = 1.00) between the spontaneous estrus and synchronized groups. The BFA values of the pregnant group (n = 34) were approximately 1.42 and 1.54 times higher than those of the nonpregnant group (n = 24) on Days 7 (0.54 ± 0.04 cm(2) vs. 0.38 ± 0.02 cm(2); P < 0.01) and 14 (0.80 ± 0.23 cm(2) vs. 0.52 ± 0.22 cm(2); P < 0.01), respectively. The TAMV of the pregnant group was approximately 1.45 times higher than that of the nonpregnant group on Day 14 (57.8 ± 3.5 cm/s vs. 40.0 ± 3.3 cm/s; P < 0.01). However, no differences were found in the CL area, CL tissue area, dominant follicle area, and plasma P4 concentrations among these groups. In addition, the best logistic regression model to predict pregnancy included scores for BFA on Day 7, BFA and TAMV on Day 14. Setting the cutoff value of BFA at 0.43 cm(2) yielded the highest sensitivity (79.4%) and specificity (75.0%) on Day 7, indicating the effectiveness of using BFA data for predicting pregnancy on Day 7. Furthermore, setting the cutoff value at one obtained from a sample with BFA 0.63 cm(2) and TAMV 50.60 cm/s yielded the highest sensitivity (85.3%) and specificity (91.7%) on Day 14. In conclusion, the evaluations of BFA on Day 7, and paired BFA and TAMV on Day 14 represent reliable predictors of pregnancy in the cow.

Development of a programmable piggyback syringe pump and four-times-a-day injection regimen for superovulation in non-lactating Holstein cows.

The objectives of the present study were to develop a programmable piggyback syringe pump for bovine superovulation and to evaluate the effects of a four-times-a-day injection regimen using the pump. Non-lactating Holstein cows were treated with a total of 30 armour units of porcine FSH by injection four times a day with the pump (study, n = 9) or injection twice a day manually (control, n = 9) for four consecutive days from D10 of the estrous cycle. The pump-driven program successfully induced superovulation in all cows tested. The numbers of small (3- < 5 mm in diameter) and large (≥ 10 mm in diameter) follicles were greater in the study group on D11-13 and D14, respectively. There were fewer unovulated follicles detected on D21 (7 days after estrus) in the study group than in the control group (1.2 ± 0.4 and 3.2 ± 0.6, respectively).

Persistence of uterine bacterial infection, and its associations with endometritis and ovarian function in postpartum dairy cows.

We investigated the relationship between the persistence of uterine bacterial infections with cytologically determined endometritis and ovarian function in 65 postpartum Holstein cows. Vaginal mucus discharges were collected, and endometrial smear samples (n = 130) were collected for cytological and bacteriological examinations from the cows at weeks 5 and 7 postpartum (pp). Blood samples were collected at weeks 3, 5 and 7 pp to determine plasma progesterone concentrations to monitor ovarian activity. According to the bacteriological examination, cows were classified into four groups. The first group (n = 32; 49%) comprised cows negative for bacteria at weeks 5 and 7 pp. The second group (n = 11; 17%) comprised cows with bacterial infections at week 5 pp but that were clear of infection at week 7 pp. The third group (n = 12; 19%) comprised cows without bacteria at week 5 pp but that acquired an infection by week 7 pp. The fourth group (n = 10; 15%) comprised cows with bacterial infections at weeks 5 and 7 pp (persistence of infection). A positive correlation (P < 0.001) was noted between the severity of cytologically determined endometritis, purulent vaginal discharge and the persistence of infection. Cows with persistent infections had a significantly (P < 0.01) prolonged luteal phase compared with cows without infection. In conclusion, the prevalence of cytologically determined endometritis and prolonged luteal phase were significantly increased in cows with persistent infections.

Effect of Postbreeding Intramuscular Administration of Deslorelin Acetate on Plasma LH and Progesterone Concentrations and Conception Rate in Mares.

To clarify effect of postbreeding intramuscular administration of deslorelin on mares, plasma LH and progesterone concentrations and conception rate were evaluated. In Experiment 1, deslorelin, buserelin, or a placebo was administered on day 9 (day 0=day of last breeding or AI). Plasma samples were collected daily from days 6 to 15, and additional samples were taken after treatment. There was a significant increase in hormonal concentrations after deslorelin treatment. In Experiment 2, deslorelin or placebo was injected on day 9. There was no difference in conception rates at days 15 and 30 between the two groups. While a single postbreeding treatment of deslorelin increased peripheral LH and progesterone concentrations, it may not affect the conception rate in mares.

Fluctuation in plasma bone metabolic markers in multiparous and primiparous Holstein cows during early to peak lactation.

Because the role of bone turnover during early to peak lactation in dairy cows is unclear, plasma levels of osteoclast- or collagen-specific bone resorption and osteoblast-specific bone formation markers from postpartum week 3 (W+3) to W+7 were compared with prepartum W-3 levels in multiparous (n = 15) and primiparous (n = 13) Holstein cows. The multiparous group showed a consistently low level of osteoclast-specific bone resorption markers, while collagen-specific resorption and osteoblast-specific formation markers decreased at postpartum W+7 and from W+5, respectively. In the primiparous group, the plasma levels of all three markers decreased from postpartum W+3. These results suggest that the calcium demand during the transition from early to peak lactation in dairy cows is less affected by bone turnover.

Mycoplasma infection in the uterus of early postpartum dairy cows and its relation to dystocia and endometritis.

This study investigated the incidence of mycoplasma infection in the uterus of postpartum Holstein dairy cows and its relationship to the occurrence of endometritis. The genital tracts of 209 cows from three dairy farms in the Iwate Prefecture, Japan, were examined at Weeks 5 and 7 postpartum. The condition of the cervicovaginal mucus was assessed using a Metricheck device and assigned a score from 0 (clear mucus) to 4 (purulent material with fetid odor). Intrauterine samples (N = 418) were collected at Weeks 5 and 7 postpartum using a cytobrush. After its withdrawal, swab samples were placed in mycoplasma culture broth at 37 °C for 72 hours. A novel and rapid polymerase chain reaction was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense). The cytobrush was also rolled gently along the length of a glass slide for subsequent polymorphonuclear neutrophil count. The diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 and 7, respectively. From a subset of cows, additional swabs were rolled against the cytobrush and then placed in transport medium. These samples were then plated on specific agar plates and cultured under aerobic and anaerobic conditions to identify other bacteria present. The incidence of dystocia at the last calving was compared in mycoplasma positive and negative cows. Of the seven mycoplasma species, only M. bovigenitalium was detected; it was detected in 31 of the 418 uterine swabs (7.4%). Twenty-four cows were positive for M. bovigenitalium (eight cows at Week 5, nine cows at Week 7, and seven cows at both Weeks 5 and 7). The incidence of dystocia was higher (P < 0.0001) in mycoplasma positive (7/24; 29.2%) compared with mycoplasma negative (4/185; 2.2%) cows. However, there was no significant association between dystocia at last calving and subsequent uterine infection with other bacteria. In addition, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (8/16; 50%) than in mycoplasma negative (47/193; 24.4%) cows at Week 7. Therefore, we concluded that M. bovigenitalium infection in the uterus might be associated with recent dystocia and with cytologic endometritis in postpartum dairy cows.

Association between energy status early postpartum and subsequent embryonic mortality in high-yielding recipient cows.

High-yielding Holstein-Friesian recipients (n = 43) were used in order to investigate the relation between energy balance status during the early postpartum period and subsequent embryonic mortality after transferring good-quality frozen embryos. Blood samples were collected during the second, third, fourth, fifth, sixth and seventh weeks postpartum in order to measure energy status indicators. These indicators include ß hydroxyl butyric acid (BHBA), non-esterified fatty acid (NEFA), total cholesterol (T-chol), glucose and blood urea nitrogen (BUN). Moreover, body condition scores (BCS) were assessed during the same period. Pregnancy diagnosis by ultrasonography at the 28th day postestrus and embryo viability was ascertained until 45 days postestrus in order to detect late embryonic mortality (LEM). The pregnancy rate on day 28 was 44.2% (19/43); however, five cows (11.6%) experienced LEM by day 45. Based on the non-return rate at day 24, non-pregnant animals, as diagnosed by ultrasonography, were allocated into animals with longer estrus intervals than 24 days (32.5%; mid-embryonic mortality (MEM) group) and animals returning to estrus by day 24 postestrus (23.5%; early embryonic mortality (EEM) group). At week 5 postpartum, BCS was significantly (P < 0.05) lower in the LEM group than that of pregnant (PREG), EEM and MEM groups. NEFA was significantly higher in animals that experienced LEM (LEM group) at week 7 postpartum (289.6 ± 47.0 µEq/L; P < 0.01) than that of PREG (196.8 ± 16.0 µEq/L), EEM (157.2 ± 18.6 µEq/L) and MEM groups (191.5 ± 14.4 µEq/L). In conclusion, lower BCS at week 5 postpartum and higher NEFAs at week 7 postpartum may be associated with subsequent LEM in high-yielding recipient cows.

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.

Validation of a direct time-resolved fluoroimmunoassay for progesterone in milk from dairy and beef cows.

The aim of this study was to validate a direct time-resolved fluoroimmunoassay (TR-FIA) for quantifying progesterone concentrations in milk during the bovine oestrous cycle. Holstein-Friesian and suckled and non-suckled Japanese Black cows were used to demonstrate the relationship between milk and plasma progesterone concentrations and to monitor progesterone profiles in milk and plasma during the oestrous cycle. The minimum detection level of the assay was 1.53ng/mL. Progesterone concentrations in milk and plasma changed in a similar manner throughout the oestrous cycle in dairy and beef cows, and milk and plasma progesterone profiles were significantly correlated (P<0.001). The study confirmed that a direct TR-FIA can be used to monitor the oestrous cycle in cattle and to quantify progesterone concentrations in whole milk.

Plasma and fecal sex steroid hormone profiles during the estrous cycle in a Japanese serow (Capricornis crispus).

We aimed to determine plasma progesterone and estradiol-17beta concentrations, as well as fecal progesterone concentrations during the estrous cycle in a female 8-year-old Japanese serow (Capricornis crispus). The step frequencies during the night were recorded by a pedometer attached to the serow's hind leg from October to the following June. Estrous behavior was also monitored during the day. Blood samples were taken once a day from 8 February to 10 March from an indwelling catheter placed in the jugular vein. Fecal samples were taken once or twice a day from 1 January to 29 April. Plasma and fecal progesterone and plasma estradiol concentrations were determined using time-resolved fluoroimmunoassays. The estrus behavior observed lasted for 2-3 days. Peak step frequencies were recorded between November and April, at intervals of 17-19 days. Plasma progesterone concentrations remained elevated (1-7.7 ng/ml) for 12 days during non-estrus, and plasma estradiol concentrations were highest when the peak step frequency and estrous behavior were observed. Step frequency increased around the times fecal progesterone levels fell to basal levels. Progesterone concentrations in feces were significantly correlated with those in sera. Thus, measurement of fecal progesterone concentrations might be useful for monitoring the reproductive status of the Japanese serow.

Effect of synchronisation of ovulation on ovarian profile and days open in holstein cows diagnosed as nonpregnant 26 days after timed artificial insemination.

The objective of this study was to clarify the effects of a second protocol of ovulation synchronisation starting on day 26 after timed artificial insemination on ovarian profile and days open in dairy cows diagnosed as nonpregnant. Ninety-four Holstein-Friesian cows received intramuscular injections of a GnRH analogue (GnRH), 100 microg fertirelin, on day 0 and a prostaglandin F(2alpha) analogue (PG), 5 mg etyprostontromethamine, on day 7. GnRH was again administered 48 h after the PG injection, and timed artificial insemination was performed 16 to 20 h later (Ovsynch/TAI). Twenty-six of the 94 cows returned to oestrus within 26 days after TAI and were inseminated. Of the other 68 cows, 44 were not pregnant and were randomly allocated to undergo another Ovsynch/TAI protocol (Resynch group; n=23) or AI only after detection of oestrus (Control group; n=21). The ovarian and hormonal profiles were compared between the first and second Ovsynch protocol periods in the Resynch group. The diameter of the dominant follicle and plasma oestradiol-17 beta concentration at the second GnRH injection were significantly greater than those at PG injection during the second Ovsynch period. Ovulation was synchronised in all of the animals in the second Ovsynch period. The AI submission rates, mean AI intervals and pregnancy rates of the Resynch and Control groups were 100% and 57.1%, 36.0 +/- 0.0 and 43.2 +/- 10.9 and 30.4% and 14.3%, respectively. The mean AI interval was 7 days shorter and the pregnancy rate was higher in the Resynch group than in the Control group, although no significant differences were found due to the small number of the animals. In conclusion, the Resynch protocol initiated on day 26 after TAI in the first protocol has the potential to reduce days open and increase the pregnancy rate in dairy cows.

A simple method for selection of cumulus-oocyte complexes from bovine ovaries by sedimentation with percoll.

Selection of bovine cumulus-oocyte complexes (COCs) for in vitro embryo production (IVP) is generally based on the morphological characteristics of the cumulus cells surrounding the oocytes and the ooplasm under microscopic observation. The purpose of this study was to examine a simple method for selection of COCs by sedimentation with Percoll solutions. COCs were aspirated from ovaries derived from a local slaughterhouse, and the COCs were classified by the morphology of their cumulus cell layers, as follows: Class A, compact and thick; Class B, compact but thin; Class C, partially denuded and thin; and Class D, denuded. Percoll solutions were prepared by diluting Percoll to 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30% solutions, respectively. COCs were placed on the surface of the Percoll solution for 3 min, and the precipitated COCs were transferred to stepwise high density solution. The percentage of Percoll solution just before buoyancy was considered to the specific sedimentary value of the COC and oocyte. The mean sedimentary value of Class A COCs was higher than those of the other classes (P<0.01). The mean sedimentary values of denuded oocytes from Classes A and B were higher than those from Classes C and D (P<0.01). Our results show that sedimentation of COCs and denuded oocytes was generally related to the morphological quality of the COCs, although the sedimentary values ranged widely for one class of COCs and oocytes. The Percoll method can be used for simple selection of COCs.

Developmental aberrations of liver gene expression in bovine fetuses derived from somatic cell nuclear transplantation.

Cloning by somatic cell nuclear transfer (NT) has been accomplished. However, the process itself is inefficient since most clones die before birth and survivors often display various anomalies. In an effort to determine global expression profiles of developmentally regulated liver genes in NT bovine fetuses, we employed a custom-made bovine liver complementary DNA (cDNA) microarray. The NT fetuses in early pregnancy were derived from cumulus cells as the nuclear donor cells. Normal fetuses were derived from in vitro fertilization (IVF) and artificial insemination (AI). Gene expression levels in NT, IVF, and AI fetal livers were obtained by comparing individual fetal liver samples with that of adult liver of nonpregnant cycling cows. Statistical analyses of the expression data showed widespread dysregulation of developmentally important genes in the three NT fetuses examined. It was found that the number of dysregulated genes was within a range of 3.5-7.7% of the tested genes in the NT fetal livers. The analyses revealed that one NT fetus was markedly different in liver gene expression profile from the other two NT fetal livers in which the expression profiles were highly correlated. Thus, our findings demonstrate that widespread dysregulation of liver genes occurs in the developing liver of NT bovine fetuses. It is possible that inappropriate genomic reprogramming after NT is a key factor associated with abnormal gene expressions in the livers of NT fetuses, whereas distinct expression patterns between the fellow cloned fetuses likely have resulted from variable epigenetic status of the donor nuclei.

Meat and milk compositions of bovine clones.

The technology is now available for commercial cloning of farm animals for food production, but is the food safe for consumers? Here, we provide data on >100 parameters that compare the composition of meat and milk from beef and dairy cattle derived from cloning to those of genetic- and breed-matched control animals from conventional reproduction. The cloned animals and the comparators were managed under the same conditions and received the same diet. The composition of the meat and milk from the clones were largely not statistically different from those of matched comparators, and all parameters examined were within the normal industry standards or previously reported values. The data generated from our match-controlled experiments provide science-based information desired by regulatory agencies to address public concerns about the safety of meat and milk from somatic animal clones.

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro.

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.