Use of equine embryo -derived mesenchymal stromal cells and their extracellular vesicles as a treatment for persistent breeding-induced endometritis in susceptible mares.

Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development may improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis, and early gestational tissue provides a unique source of multipotent stem cells for creating MSCs. Extracellular vesicles (EVs) are mediators of cell communication produced by many different cell types. This study utilized embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in two groups: a) PBIE-susceptible mares challenged with pooled dead sperm (n=5); and b) client-owned mares diagnosed as susceptible to PBIE (n=37 mares and 40 estrous cycles). Mares pre-treated with intrauterine EDMSCs or their EVs resulted in a significant reduction in the accumulation of intrauterine fluid post-breeding. Nine of 19 (47 %) mares treated with EDMSCs prior to natural breeding and 13 of 20 (65 %) mares treated with EDMSC derived EVs were pregnant after the first cycle and 12 of 18 (67 %) mares treated with EDMSCs, and 15 of 19 (79 %) mares treated with EVs conceived by the end of the breeding season. These preliminary clinical studies are the first reports of the use of EDMSCs or their EVs as a potential intrauterine therapy for the management of PBIE susceptible mares.

Factors influencing parentage ratio in canine dual-sired litters.

Breeding a bitch with two different sires during a single estrous cycle has the potential to facilitate rapid genetic gain and improve reproductive performance within a canine breeding colony. There is limited data regarding the factors that contribute to the success of dual-sired litters in domestic dogs, and only anecdotal evidence suggesting that these litters rarely produce offspring from more than one sire. The objective of this prospective clinical study was to investigate multiple factors that likely affect the success of dual-sired litters on whelping rate, litter size and parentage ratio. These factors include: timing of artificial insemination (AI), order of sires, number of AI's per cycle, semen type, sperm quality and age of sire and bitch. Data collected over a 10 year period from twenty-nine estrous cycles (28 individual bitches of 10 different breeds) were evaluated after an initial AI with frozen semen from the 'genetically desired' sire and followed up with a second AI with either fresh (n = 9) or frozen (n = 16) semen or natural mating (n = 4) from a different, 'back up', sire. DNA parentage of each pup born was determined by using a primary panel of 288 SNPs. The whelping rate and litter size from previous single sire inseminations per estrous cycle, in the same bitches, (n = 16) over 25 estrous cycles using either fresh (n = 4) or frozen-thawed (n = 21) semen, were analyzed as controls. Of the 29 dual-sired breedings, 26 bitches whelped (89.7%), and 8 litters (30.8%) were of mixed parentage. In the litters of mixed parentage after a dual-sired breeding, a greater proportion of the offspring were from the second sire than the first sire (73.0% and 27.0% respectively; P < 0.05). Interestingly, in litters where all pups were of single sire parentage after a dual-sired breeding, 50.0% of the offspring were by the first sire and 50.0% were by the second sire. For litters of mixed or single paternity produced by dual-sired breeding there was no difference in average litter size. However, on a per estrous cycle basis for each bitch the whelping rate (89.7% v. 76.0%.) and litter size (5.5 ± 2.5 v. 4.0 ± 2.78) of all dual-sired breedings were greater (P < 0.05) compared to previous single-sired breedings (controls) respectively. This study demonstrates that offspring of mixed parentage derived from dual-sired breedings may be achieved. Furthermore, insemination with semen from two different sires may increase the whelping rate and litter size, which is an important consideration when using genetically valuable, or older individuals with potentially reduced fertility.

Factors affecting the reproductive performance of bitches: A prospective cohort study involving 1203 inseminations with fresh and frozen semen.

The aim of this prospective cohort study was to utilize multivariable statistical methods to identify factors that significantly affected whelping rate, litter size and gestation length in a large population of bitches of many different breeds, presented for routine breeding management. In addition, we aimed to determine the incidence of dystocia and the proportion of bitches undergoing a caesarean section procedure. A total of 1146 individual bitches representing 84 different breeds contributed 1203 inseminations over the 9 year (2007-2015) study period. Bitches were inseminated with either frozen-thawed (n = 645), fresh (n = 543) or chilled (n = 15) semen from 1371 different males. The mean (SD) whelping rate was 74± 4% and the mean litter size was 5.8 ± 3.1 pups per litter for all bitches in the study. The whelping rate was significantly lower in bitches inseminated with frozen-thawed semen compared with bitches inseminated with fresh semen (71% vs 80% respectively; P < 0.001). Semen that was classified as having poor motility (<30% progressive) resulted in a significantly lower whelping rate (37%) than semen classified as good (30-65% progressive; whelping rate = 67%) or excellent (>65% progressive; whelping rate = 79%). There was a linear decline in whelping rate with advancing age. Greyhounds and Labradors demonstrated a significantly higher whelping rate (88% and 94% respectively) compared with all other breeds (71.3%, P < 0.001). Bitches inseminated with frozen-thawed semen had significantly smaller litter sizes than bitches inseminated with fresh semen (5.4 ± 3.1 vs 6.2 ± 3.0 pups per litter respectively; P = 0.02). Smaller breeds had significantly smaller litters (4.4 ± 2.1 pups) than medium (5.2 ± 2.9 pups), large (5.9 ± 2.9 pups) or giant (6.7 ± 3.8 pups) breeds. For each advancing year of age, litter size decreased by 0.13 pups per litter. The mean (SD) gestation length from LH0 was 65 ± 1.9 d. Greyhounds had a significantly longer pregnancy duration (68.0 ± 1.5 d) than other breeds. For each additional year of bitch age, gestation length increased by 0.11 days (P < 0.01), and for each additional pup per litter, gestation length was reduced by 0.08 days (P < 0.05). Of the 890 bitches for which whelping outcomes were recorded; 409 (46%) whelped normally without assistance, 249 (28%) had an elective C-section, 205 (23%) underwent an emergency C-section and 27 (3%) were medically managed or required veterinary assistance for dystocia. Brachycephalic breeds were 11.3 (95CI = 9.3-17.9; P < 0.001) times more likely to have a C-section compared to all other breeds. Bitches with litter sizes of one or two pups had a C-section rate of 83%, whereas bitches with litter sizes of three or more pups had a C-section rate of 43% (P < 0.001). This study provides important clinical information to optimise whelping rates, litter size and the prediction of whelping in certain breeds for clinicians working in canine reproduction.

Determination of the normal reference interval for anti-Müllerian hormone (AMH) in bitches and use of AMH as a potential predictor of litter size.

Anti-Müllerian hormone (AMH) is a reliable endocrine marker of ovarian reserve in many species with extensive literature in both humans and cattle. However, there are no known hormonal predictors of ovarian reserve and potential reproductive performance in the bitch. A prospective cohort study was performed involving 155 intact bitches of various ages (range 1.2-7.6 years) and breeds that were presented for routine breeding management over a one-year period. All bitches were artificially inseminated with frozen or fresh semen using the transcervical insemination (TCI) technique. AMH concentrations were measured using a commercially available canine AMH ELISA (Ansh Labs® , Texas, USA), which we validated prior to performing the study. The reference interval (RI) for AMH for all bitches in the study, regardless of body weight, was 2.9-21.1 ng/ml. There was a significant effect of bitch size and age on AMH concentrations. The RI for giant breeds was significantly (p < .01) lower (1.75-15.6 ng/ml) than small-sized (5.6-24.2 ng/ml), medium-sized (4.3-23.7 ng/ml) and large-sized (4.3-21.0 ng/ml) bitches. The mean AMH concentration in bitches less than 4 years of age was 12.4 ng/ml, whereas the mean AMH concentration in bitches older than 4 years of age was 10.5 ng/ml (p < .05). For each additional year of age above 1 year of age, AMH concentrations fell by 0.5 ng/ml. There was no effect of AMH concentration on the whelping rate. Smaller breeds had smaller litters (and higher AMH concentrations), but within each breed size category, bitches with higher AMH concentrations had significantly larger litter sizes (p < .01). For each 1 ng/ml increase in AMH, litter size increased by 0.3 pups/litter. In conclusion, we determined a normal reference interval for AMH in bitches based on body weight using a canine-specific assay. In agreement with findings in humans and other species, we found that there is a decline in AMH concentrations with advancing age in bitches. Finally, the significant positive correlation between AMH concentrations and litter size indicates that AMH may be a useful management tool for the selection of bitches in breeding programmes.

Reproductive performance of Thoroughbred mares in the Waikato region of New Zealand: 2. Multivariable analyses and sources of variation at the mare, stallion and stud farm level.

AIM: The objective of this study was to utilise multivariable statistical methods appropriate for clustered data to identify mare-related explanatory variables that significantly affected the reproductive performance of Thoroughbred mares in the Waikato region of New Zealand. In addition, we aimed to determine the relative contribution of the mare, stallion and stud farm to reproductive performance. METHODS: A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during three consecutive breeding seasons (2006-2008). A total of 1,482 individual mares contributed 2007 mare years and 3,402 oestrous cycles over the three breeding seasons. Reproductive performance was measured using three parameters; (a) first-cycle pregnancy rate (FCPR), (b) end-of-season pregnancy rate (SPR), and (c) the start-of-mating to conception interval. RESULTS: When controlled for the effects of serving stallion, stud farm and year of study the only significant mare-related variables included in the final models of FCPR, SPR and conception interval were the age of the mare and her reproductive status (classified as dry or foaling). Advancing mare age significantly reduced reproductive performance regardless of reproductive status and foaling mares had significantly poorer reproductive outcomes compared with dry mares when controlled for age. For each additional increase in year of age, the FCPR was reduced by a factor of 0.94 (95% CI=0.92-0.96) and the SPR was reduced by a factor of 0.91 (95% CI=0.88-0.93). Mares older than 14 years of age took longer to conceive after the start-of-mating compared with younger mares. The daily hazard of conception for mares 14 years and older was 0.64 (95% CI=0.47-0.83) times less than mares younger than 9 years of age. Determining the relative contribution of the mare, stallion and stud farm to the FCPR indicated that 95.9% of the variation was at the mare level, 4.1% was at the stallion level and 0% was at the stud farm level. For the SPR the variance components indicated that 92.5% of the variation was at the mare level, 6.7% was at the stallion level and 0.8% was at the stud farm level. CONCLUSIONS: The reproductive performance of Thoroughbred mares in the Waikato region of New Zealand is influenced by two main mare-related factors; the age of the mare and her reproductive status (dry or foaling). The majority of variation in reproductive performance was associated with mare-level factors and the contribution of the stallion and stud farm was relatively minor.

Reproductive performance of Thoroughbred mares in the Waikato region of New Zealand: 1. Descriptive analyses.

AIM: To describe the reproductive performance of a population of Thoroughbred mares on stud farms in the Waikato region of New Zealand. METHODS: A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during three consecutive breeding seasons (2006-2008). A total of 1,482 individual mares contributed 2007 mare years and 3,402 oestrous cycles over the three breeding seasons. Mares were served by 87 individual stallions. RESULTS: The mean first-cycle pregnancy rate (FCPR) was 53.6%, the end-of-season pregnancy rate (SPR) was 85.3% and the foaling rate was 80.2%. There were significant differences amongst farms for FCPR (range 47.3-68.2%; p<0.01), SPR (range 81.4-92.6%; p<0.001) and foaling rate (range 77.8-90.1%; p<0.001). There were significant differences amongst stallions in their FCPR (range 38-74%; p<0.001) and SPR (range 72-97%; p<0.001).The proportion of pregnancies lost between day 14-16 and day 42 of gestation was 5.5% and the proportion of pregnancies lost between day 42 and term was 3.0%. Mares older than 14 years of age and barren mares had the highest pregnancy loss rates. Of all pregnancies diagnosed at 14-16 days, 12.9% were multiple pregnancies. The length of the breeding season was relatively short with 87.3% of services occurring in the 91-day period between 1 September and 30 November. CONCLUSIONS: The reproductive performance of Thoroughbred mares in the Waikato region of New Zealand was similar to that reported from other major Thoroughbred breeding countries. Although the per-cycle pregnancy rates in this study were lower than those reported in some studies, the foaling rate was similar to previous reports and this was achieved during a relatively short breeding season.

The reproductive performance of Thoroughbred mares treated with intravaginal progesterone at the start of the breeding season.

The objective of this study was to investigate the effects of intravaginal progesterone on the reproductive performance of transitional Thoroughbred mares on commercial stud farms. Two hundred twenty-seven (227) non-lactating transitional Thoroughbred mares aged between 4 and 18 y (mean 9.4 ± 3.2 y) located on three stud farms in the Waikato region of New Zealand were used in the study performed during four consecutive breeding seasons (2007-10). Mares were age-matched in pairs and either treated with an intravaginal progesterone releasing device (Cue-Mare, 1.72 g progesterone, 10% w/w) for up to 10 d (Treated; n = 126) or left untreated (Control; n = 101). In both groups, 1,667 iu of hCG was given IV when an ovarian follicle ≥35 mm was detected (in conjunction with estrous behavior) and each mare was bred by natural service. Treated mares were served earlier in the breeding season (mean ± SD interval to first service was 13.9 ± 3.0 vs 26.7 ± 13.2 d for Treated and Control groups, respectively; P < 0.001). In the Treated and Control groups, 95.2 and 42.6% of mares were served within the first 21 d of the season (P < 0.001). Treated mares conceived earlier in the breeding season (mean number of days to conception 37.5 ± 14.2 vs 50.8 ± 21.3 d, P = 0.01). There was no difference between groups in the first service pregnancy rates (53.9 and 50.5% for Treated and Control mares, P = 0.89). Treatment with an intravaginal device increased the number of mares conceiving by the end of the breeding season (91.3 vs 82.3% for Treated and Control groups, P = 0.04). Therefore, this treatment protocol appeared to offer a convenient, economical and reliable method for managing transitional mares on commercial Thoroughbred stud farms.

Calcium, parathyroid hormone, oxytocin and pH profiles in the whelping bitch.

Despite the high prevalence of primary uterine inertia in whelping bitches, the underlying pathogenesis remains unclear. The objectives were to i) determine serum concentrations of total calcium, ionized calcium (iCa), parathyroid hormone (PTH), and blood pH in normally whelping bitches throughout the peri-parturient period; and ii) investigate relationships among iCa, PTH, and acid-base status, and the role that they and oxytocin may have in the underlying pathogenesis of canine uterine inertia. Bitches were randomly selected from a population of German Shepherd Dog bitches with a history of uncomplicated parturition (Group 1; n=10), and from a population of Labrador bitches with a clinical history of an increased incidence of uterine inertia and stillbirths (Group 2; n=20). Jugular blood samples were collected daily from -4 d to the onset of whelping (t=0 h), and then every 4h until the last pup was born. Overall, bitches from Group 2 had higher mean+/-SEM serum concentrations of PTH (4.72+/-2.45 pmol/L, P<0.001), lower iCa (1.31+/-0.08 pmol/L, P<0.05), and higher venous pH (7.41+/-0.03, P<0.005) than bitches from Group 1 (2.9+/-1.44 pmol/L, 1.38+/-0.06 mmol/L, and 7.33+/-0.02, respectively) during the periparturient period. However, there was no significant difference between Groups 1 and 2 for serum oxytocin concentrations during the periparturient period (45.5+/-40 and 65.5+/-82 pg/mL). We inferred that low iCa resulting from a rising pH and decreasing PTH during the periparturient period may have contributed to decreased uterine contractility and increased risk of stillbirths. Therefore, manipulating the cationic/anionic difference in diets of pregnant bitches, similar to the bovine model for hypocalcamia, may reduce the incidence of stillbirths in the bitch.

The effect of hCG administration five days after insemination on the first service conception rate of anestrous dairy cows.

The aim of this study was to investigate the effect of treating anovulatory anestrous (AA) dairy cows with 1500 IU of hCG IM, 5 d after insemination, on their first service conception rate. A clinical trial was conducted during the 2003/2004 breeding season involving 442 AA dairy cows in six herds. On Day -8, all cows were treated with a progesterone-containing intravaginal device (Cue-Mate). The devices were removed on Day -2, and on Day -1 all cows received an IM injection of 1mg of estradiol benzoate. Cows in the control group (n=220) received no further treatments. Cows in the treatment group (n=222) which had been inseminated on Days 0 or 1 were treated with 1500 IU of hCG IM 5 d after insemination. Blood was collected from 30 cows (15 in each group) on Days 5 and 12 after AI for analysis of plasma P4 concentration. There was no difference in first service conception rates between the control and treatment groups (46.3% versus 43.6%, respectively; P=0.68), despite the fact that plasma P4 concentrations were higher in the treatment group on Day 12 (4.9+/-1.3 ng/mL versus 6.2+/-2.7 ng/mL for control and treatment groups, respectively; P<0.01). In conclusion, 1500 IU of hCG 5 d after insemination did not improve first service conception rate in AA dairy cows.

Reproductive performance in anestrous dairy cows following treatment with two protocols and two doses of progesterone.

The objectives of this study were to evaluate the effect using two doses of progesterone (P4) releasing devices in two different programs on reproductive performance of anestrous dairy cows. Cows (n = 1555) not detected in estrus by 10 d before the planned start of the seasonal breeding program and in which no CL was palpable were treated with an intravaginal P4-releasing device ('Single'; approximately 1.56 g of P4) or a modified device with triple the normal P4 dose ('Triple'; approximately 4.7 g of P4). The devices were in place for either 6 d ('Short') or 8 d ('Long'), with 1mg estradiol benzoate (EB) given 24 h after device removal. The 'Long' program also included treatment with 2 mg EB at device insertion. The Long program resulted in a higher first service conception rate (RR = 1.18 (95% CI = 1.03-1.33); P = 0.02), but had no effect on the 28-d, 56-d or final pregnancy rate compared to the Short program. There were no effects of dose of P4 on any outcome. In conclusion, the Long compared to the Short program, but not the dose of P4, improved first service conception rates in anestrous cows.

Supplementing previously treated anestrous dairy cows with progesterone does not increase first-service conception rate.

The objective of this study was to investigate the effect of supplementing previously treated anovulatory anestrous (AA) dairy cows with progesterone delivered intra-vaginally for 7 days, commencing 4 or 5 days after insemination, on first-service conception rate. A clinical trial, involving 990 AA dairy cows in 14 dairy herds, was conducted during the 2002/2003 breeding season. On Day -8, all cows were treated with a progesterone-containing intravaginal device (Cue-Mate). The devices were removed on Day -2; on Day -1, all cows were given 1mg of estradiol benzoate im. Cows in the control group (n = 499) received no further treatments. Cows in the treatment group (n = 491) that had been inseminated on Day 0 or 1 had a new device inserted (on Day 4 or 5), with removal of the device after 7 days. First-service conception rates for the control and treatment groups were not different (35.0% versus 36.7% respectively; P = 0.41). Furthermore, there was no difference in conception rates between cows that had devices inserted on Day 4 or 5 (31.3% versus 37.2% respectively; P = 0.45). In conclusion, supplementation of previously treated AA dairy cows with an intravaginal progesterone-releasing device for 7 days (commencing 4 or 5 days after insemination) did not significantly improve first-service conception rate.

Immunodetection of Parkin protein in vertebrate and invertebrate brains: a comparative study using specific antibodies.

Parkin is an intracellular protein that plays a significant role in the etiopathogenesis of autosomal recessive juvenile parkinsonism. Using immunoblot methods, we found Parkin isoforms varying from 54 to 58 kDa in rat, mouse, bird, frog and fruit-fly brains. Immunocytochemical studies carried out in rats, mice and birds demonstrated multiple cell types bearing the phenotype for Parkin throughout telencephalic, diencephalic, mesencephalic and metencephalic brain structures. While in some instances Parkin-containing neurons tended to be grouped into clusters, the majority of these labeled nerve cells were widely scattered throughout the neuraxis. The topographical distribution and organizational pattern of Parkin within major functional brain circuits was comparable in both rats and mice. However, the subcellular localization of Parkin was found to vary significantly as a function of antibody reactivity. A consistent cytoplasmic labeling for Parkin was observed in rodent tissue incubated with a polyclonal antibody raised against the human Parkin protein and having an identical amino-acid sequence with that of the rat. In contrast, rodent tissue alternately incubated with a polyclonal antibody raised against a different region of the same human Parkin protein but having 10 mismatched amino-acid sequence changes with those of the rat and mouse, resulted in nuclear labeling for Parkin in rat but not mouse neurons. This difference in epitope recognition, however, was reversed when mouse brain tissue was heated at 80 degrees C, apparently unmasking target epitopes against which the antisera were directed. Collectively, these results show a high degree of conservation in the cellular identity of Parkin in animals as different as drosophilids and mammals and points to the possibility that the biochemical specificities of Parkin, including analogous functional roles, may have been conserved during the course of evolution.

The reproductive performance of anoestrous dairy cows following treatment with progesterone and oestradiol prior to the start of mating.

AIMS: To determine the reproductive performance of cows diagnosed as anoestrus prior to the planned start of mating (PSM) when they were either treated when first diagnosed, or left untreated until 16 days after the PSM. METHODS: A clinical trial was conducted during the 1996/97 and 1997/98 breeding seasons involving 823 anoestrous dairy cows in 14 herds. On Day -8 (PSM = Day 0), cows in one group (Treated) were each treated with an intravaginal device containing 1.9 g of progesterone (CIDR). The CIDR device was removed on Day -2, and on Day -1 each cow was injected intramuscularly with 1 mg oestradiol benzoate. Cows in the second group (Control) remained untreated at the time of first examination. All cows detected in oestrus after the PSM were mated by artificial insemination (AI) or a bull. Sixteen days after the PSM, all cows that had not been mated were presented for veterinary examination, and those which were still classified as anoestrus were treated with the previously described CIDR regimen. Pregnancy status and approximate date of conception were determined by palpation per rectum 10 -13 weeks after the PSM or 6 weeks after the end of the mating period. RESULTS: Treatment of anoestrous cows 8 days before the PSM significantly increased the number of cows detected in oestrus (95.0% vs 63.1%; p<0.001) and conceiving (59.5% vs 38.8%; p<0.001) during the first 21 days of mating, and reduced the interval from PSM to conception by 7.5 days (p<0.001). There was no significant difference between the conception rate of cows mated following the CIDR treatment regimen compared to cows mated at their first spontaneous oestrus after calving (52.4% vs 58.3%; p = 0.143). CONCLUSION: Diagnosis and treatment of anoestrous dairy cows prior to the start of mating significantly improves their reproductive performance under the seasonal mating conditions typical of spring-calving New Zealand dairy herds.

The effects of calcium supplementation of dairy cattle after calving on milk, milk fat and protein production, and fertility.

AIM: To determine the effect of calcium supplementation of dairy cattle after calving on serum concentrations of calcium, magnesium, and phosphorous after calving, and on reproductive and productive performance in the following lactation. METHODS: This was a double-blind randomised clinical trial using 356 dairy cows of parity 3 or greater from six seasonally calving commercial dairy herds. Cows were randomly assigned to a treatment and control group. The treatment group received two doses of 380 ml of calcium chloride in oil (49 g of elemental calcium); the first dose was given as soon as practical after calving and the second 12 hours later. Control cows received 380 ml of a placebo using the same treatment regime. Treatment and control cows had blood samples taken during the first 7 days after calving to determine serum calcium, magnesium and phosphorous levels. Treatment and control cows were monitored throughout the lactation to measure production and reproductive performance. RESULTS: Treatment had no effect on PSM to first service intervals. Treatment significantly reduced PSM to conception intervals in only one of the four herds with data available for analysis (p = 0.09). There was no difference between treatment groups for adjusted corrected milk yield calculated from the first herd test milk yield (p = 0.93) or 305-day milk solids production (p = 0.19). CONCLUSION: Further studies are required to identify reasons for the between-herd effectiveness of calcium chloride treatment in improving fertility.

Identification, partial characterization, and genetic mapping of kinesin-like protein genes in mouse.

Microtubule-dependent motors of the kinesin superfamily have undergone structural and functional diversification during evolution and play crucial roles in cell division and intracellular transport. Degenerate oligonucleotides homologous to highly conserved regions of sequence within the motor domain were used in a polymerase chain reaction to isolate five new members (KIF3C, KIFC2, KIFC3, KIFC4, and KIF22) of the kinesin superfamily from a mouse brain cDNA library. Northern analysis showed that KIF3C and KIFC2 are expressed mainly in neural tissues, that KIFC4 and KIF22 are expressed primarily in proliferative tissues and cell lines, and that KIFC3 is apparently ubiquitous. To elucidate the organization of genes encoding kinesin-like motors in the mouse genome and to explore the potential associations of these genes with classical mouse mutations or human genetic diseases, these new genes as well as genes encoding the previously reported KIF3A and KIF3B motors were mapped to mouse chromosomes by using an interspecific backcross panel of DNAs from The Jackson Laboratory. The data indicate that the gene KIFC4 is present in three copies in the mouse genome on chromosomes 13 (KIFC4A), 10 (KIFC4B), and 17 (KIFC4C). The gene KIF22 is present in two copies on chromosomes 7 (KIF22A) and 1 (KIF22B). The genes KIF3A, KIF3B, KIF3C, KIFC2, and KIFC3 are each single loci and map to chromosomes 11, 2, 12, 15, and 8, respectively.

Ovulatory responses and plasma luteinizing hormone concentrations in dairy heifers after treatment with exogenous progesterone and estradiol benzoate.

The objectives of this experiment were to determine the effects of 0.5 mg estradiol benzoate, administered intramuscularly 24 h after removal of CIDR-B progesterone containing intravaginal devices, on the time to estrus, ovulation and peak LH concentration in dairy heifers. Ovulatory responses and plasma LH concentrations were examined using 14 Friesian dairy heifers in 2 separate treatment periods. All heifers received a CIDR-B progesterone-containing intravaginal device with an attached 10-mg estradiol benzoate capsule for 12 d. Within each period, 24 h after CIDR-B removal, 7 heifers received an intramuscular injection of 0.5 mg estradiol benzoate while the remaining 7 heifers received an intramuscular injection of a placebo. Blood samples for LH assay were collected at 0, 6 and 12 h, and then every 4 h for 60 h after estradiol injection. Detection of estrus was conducted at 4-h intervals, and ultrasonographical examination to detect ovulation was conducted every 8 h for 88 h after removal of the CIDR-B device. Treatment with estradiol benzoate tended to reduce the time from device removal to the LH peak in Period 1 (median time to LH peak 40.1 vs 63.9 h; P = 6.07). In Period 2, treatment with estradiol had no significant effect on the time to the LH peak, standing estrus or ovulation. We hypothesize that the period effect was due to the stage of cycle at the time of treatment. For heifers treated in Period 1, the stage of cycle was random. However, because of the prior synchronization of estrus, which was implicit in the experimental design, heifers in Period 2 tended to be in late diestrus. The administration of estradiol benzoate after treatment with exogenous progesterone appears to overcome the variability in timing of LH peaks typically occurring in a herd of synchronized heifers due to different stages of follicular development.

Characterization of KIFC2, a neuronal kinesin superfamily member in mouse.

Members of the kinesin superfamily of microtubule-associated proteins are involved in a variety of intracellular processes including cell division and organelle transport. In the case of axonal transport, all kinesin superfamily members reported thus far appear to play a role in anterograde transport, while a different type of microtubule motor, dynein, appears to function in retrograde transport. To better understand the role of kinesins in axonal transport, we cloned and characterized KIFC2, a novel kinesin superfamily member from mouse brain. KIFC2 encodes a 792 amino acid protein, which contains the conserved motor domain at the C-terminal end of the protein and is most similar to members of the KAR3 family involved in cell division. However, expression analysis localized KIFC2 mRNA to nonproliferative neuronal cells in the central nervous system, and immunolocalization studies demonstrated that KIFC2 is present in axons and dendrites of neurons in the central and peripheral nervous systems. Immunolocalization and biochemical fractionation studies suggest that KIFC2 localizes with some, but not all, axonally transported organelles. Finally, ligation of mouse peripheral nerves showed that KIFC2 accumulates at the proximal and distal sides of an axonal ligature. Taken together, the data suggest that, unlike other C-terminal motor proteins that appear to be involved in cell division, KIFC2 may play a role in retrograde axonal transport.

Re-insertion of a progesterone-containing intravaginal device to synchronise returns to oestrus in dairy heifers.

Recommendations for oestrus synchronisation of dairy heifers using progesterone-containing intravaginal devices suggest re-insertion of used devices 16 days after first insemination for a period of 5 days to allow a second opportunity for artificial insemination. Controlled studies on the effectiveness of re-using intravaginal devices to synchronise returns to oestrus in non-pregnant dairy heifers are lacking. A clinical trial was conducted involving 750 Friesian heifers in 13 herds. After an initial synchronisation programme, the used intravaginal devices were re-inserted 14 or 16 days after first insemination into half of the heifers in each herd for a period of 5 days. After the first synchronisation programme, 47.5% of heifers remained non-pregnant. Re-insertion of used intravaginal devices for 5 days significantly increased the number of non-pregnant heifers detected in oestrus and inseminated by 48 hours after device removal compared to heifers in which devices were not re-inserted (45.2% v. 27.3%, p < 0.05, in herds where intravaginal devices were re-inserted on day 14; 48.8% v. 13.6%, p < 0.05, in herds where intravaginal devices were re-inserted on day 16). Re-insertion at 14 or 16 days after first insemination was equally effective in increasing visible returns to service. However, the number of non-pregnant heifers synchronised for a second round of artificial insemination was less than expected. Conception rate to the re-synchronised oestrus was unaffected by the treatment. It is concluded that the additional procedures of CIDR re-insertion, removal, tailpainting and insemination involved in there-synchrony programme, and the relatively low in-calf rate to the re-synchronised round of insemination, reduced the potential benefits of re-synchronisation.

The pharmacokinetics of salicylate in dairy cattle are not altered by simultaneous intravenous ceftiofur sodium and DL-lysine-acetyl salicylate (aspirin).

This study evaluated potential alterations to the pharmacokinetics of salicylate by concurrently administered ceftiofur sodium. The trial design was a cross-over using 10 non-lactating, non-pregnant dairy cows. In the first period each cow received intravenously (i.v.) 26 mg/kg of DL-lysine acetyl salicylate (aspirin) followed immediately by 2 mg/kg ceftiofur sodium. In the second period each cow received 26 mg/kg of aspirin i.v. Plasma samples were harvested for determination of salicylate concentration by HPLC. The data best fitted a single compartment open model, using weighted non-linear regression. No alterations to the pharmacokinetic parameters of salicylate in cattle by concurrently administered ceftiofur sodium were detected (P < 0.05). Using 90% confidence intervals, and testing for changes of > 20%, control values, elimination half-life (t1/2), apparent volume of distribution (Vd), area under the plasma concentration versus time curve (AUC) and mean residence time (MRT) were not altered. For control animals the elimination rate constant (k(el)) and total body clearance (Cl) were 1.35 +/- 0.43 h-1 and 20.2 +/- 6.1 ml/h.kg respectively (mean +/- SD). Since ceftiofur sodium did not affect the pharmacokinetics of salicylate, dose regimens for aspirin in cattle need not be altered when ceftiofur sodium is administered concurrently.