Artificial insemination of cattle: Description and assessment of a training program for veterinary students.

The objective was to assess the effectiveness of a comprehensive artificial insemination (AI) training program designed to facilitate an understanding of the breadth of the AI process, including AI skill acquisition, for preclinical veterinary students. Participants (n = 303) were enrolled at the Ross University School of Veterinary Medicine (Basseterre, St. Kitts, West Indies). The 2-d AI training program (n = 20) consisted of ∼8 h of instruction and ∼8 h of demonstration and hands-on activity. Oral presentations were used to deliver educational content, followed by video clips, discussion, demonstrations, and hands-on activity. Reproductive anatomy and physiology of the estrous cycle, AI sire acquisition, collection, evaluation, cryopreservation and distribution of conventional and sexed semen, storage and handling of frozen semen, use of synchronization protocols, accurate and efficient detection of estrus, and correct AI technique were discussed. True or false pre- and posttests were used to determine the level of knowledge gained by participants during the AI training program. Preclinical veterinary students were required to complete a semen handling and AI technique practical exam to achieve a certificate of completion. Participant program evaluations conducted at the conclusion of the program indicated that veterinary students found the content, structure, discussion, demonstrations, and hands-on activities to be appropriate and useful. No negative comments were offered about the training program, instructor, or activity coinstructors. The AI training program increased the posttest knowledge scores of veterinary students by 22 percentage points. Only 1 participant was unable to achieve a certificate of completion due to failure of the semen handling and AI technique practical exam. These results provide evidence that the AI training program was relevant and effective and that it offered information and skill acquisition with immediate field application.

Short communication: Reproduction outcomes in dairy heifers following a 14-d progesterone insert presynchronization protocol.

The objectives were to evaluate pregnancy per artificial insemination (AI), days to first AI, and proportion pregnant within 7 d of AI eligibility in dairy heifers subjected to presynchronization compared with dairy heifers not presynchronized. Thirty days before AI eligibility, Holstein heifers were assigned randomly to 1 of 3 groups: 14-d controlled internal drug release (CIDR; containing progesterone) presynchronization, PGF2α presynchronization, or control (no presynchronization). Heifers in the 14-d CIDR presynchronization treatment (n = 119) received a CIDR on d -30, which was removed on d -16, followed by an injection of PGF2α upon entry to the breeding program (d 0). Heifers in the PGF2α presynchronization treatment (n = 118) received an injection of PGF2α on d -11 and d 0. Control heifers (n = 121) were not presynchronized and received an injection of PGF2α on d 0. All heifers received tail paint on d 0 to facilitate once-daily detection of estrus (based on paint removal). Heifers detected in estrus received AI with conventional semen on the same morning as detected estrus. Generalized linear mixed models were used to assess mean treatment differences. Following PGF2α treatment on d 0, more heifers were detected in estrus in the first 7 d after eligibility in the 14-d CIDR group (95.8%) compared with the PGF2α (74.6%) and control (66.9%) groups. Days to first AI differed between treatments (14-d CIDR = 3.6 d vs. PGF2α = 5.0 d vs. control = 6.8 d). Pregnancy per AI for first AI within 7 d of eligibility was 71.9% (14-d CIDR), 58.0% (PGF2α), and 61.7% (control), and differed between 14-d CIDR and PGF2α heifers. Presynchronization with a 14-d CIDR increased the proportion of heifers pregnant in the first 7 d of eligibility (14-d CIDR = 68.9% vs. PGF2α = 43.2% vs. control = 41.3%). Projected days on feed (d 0 to projected calving date) were 295 (14-d CIDR), 302 (PGF2α), and 305 (control), and were different between the 14-d CIDR and control heifers. The potential economic benefit to the producer was $15.85 per heifer presynchronized with a 14-d CIDR protocol compared with the control group. Treatment of dairy heifers with a 14-d CIDR effectively presynchronized estrus, resulting in a greater proportion detected in estrus, reduced days to first AI, and an increased proportion of heifers pregnant within the first 7 d after breeding eligibility compared with heifers presynchronized with a single PGF2α injection and control heifers.

Genome-wide association analysis and gene set enrichment analysis with SNP data identify genes associated with 305-day milk yield in Holstein dairy cows.

Milk production traits, such as 305-day milk yield (305MY), have been under direct selection to improve production in dairy cows. Over the past 50 years, the average milk yield has nearly doubled, and over 56% of the increase is attributable to genetic improvement. As such, additional improvements in milk yield are still possible as new loci are identified. The objectives of this study were to detect SNPs and gene sets associated with 305MY in order to identify new candidate genes contributing to variation in milk production. A population of 781 primiparous Holstein cows from six central Washington dairies with records of 305MY and energy corrected milk were used to perform a genome-wide association analysis (GWAA) using the Illumina BovineHD BeadChip (777 962 SNPs) to identify QTL associated with 305MY (P < 1.0 × 10-5 ). A gene set enrichment analysis with SNP data (GSEA-SNP) was performed to identify gene sets (normalized enrichment score > 3.0) and leading edge genes (LEGs) influencing 305MY. The GWAA identified three QTL comprising 34 SNPs and 30 positional candidate genes. In the GSEA-SNP, five gene sets with 58 unique and 24 shared LEGs contributed to 305MY. Identification of QTL and LEGs associated with 305MY can provide additional targets for genomic selection to continue to improve 305MY in dairy cattle.

Evaluation of reproductive and economic outcomes of dairy heifers inseminated at induced estrus or at fixed time after a 5-day or 7-day progesterone insert-based ovulation synchronization protocol.

The objectives of the current experiment were to evaluate the reproductive performance and economic outcome of 3 synchronization strategies for first artificial insemination (AI) of dairy heifers. Holstein heifers from 2 herds (site A, California, n=415; site B, Idaho, n=425) were assigned to 1 of 3 treatments. Heifers assigned to the AI on estrus (AIE) treatment received an injection of 25mg of PGF(2α) at enrollment (d 0) and every 11 d thereafter until AI occurred. Heifers assigned to the CIDR5 treatment received a controlled internal drug release insert (CIDR) containing 1.38 g of progesterone, which was removed 5 d later concomitantly with an injection of 25mg of PGF(2α), and received fixed-time AI (TAI) concomitantly with an injection of 100 µg of GnRH 53 to 60 h later. Heifers assigned to the CIDR7 treatment received a CIDR insert, which was removed 7 d later concomitantly with an injection of 25mg of PGF(2α), and received TAI concomitantly with an injection of 100 µg of GnRH 53 to 60 h later. Heifers were observed for estrus and inseminated up to 98 and 73 d after enrollment in sites A and B, respectively. Thereafter, heifers were moved to pens with bulls and considered failure to conceive to AI if still not pregnant at the end of the observation period. Economic outcomes were based on cost of synchronization protocol (CIDR treatment=$11, PGF(2α) or GnRH treatments=$2.5/treatment, estrous detection=$0.80/heifer per day), rearing cost ($2.75/heifer per day), and economic loss if a heifer did not conceive to first AI ($150). Input cost of the reproductive programs=synchronization protocol cost + semen cost + rearing cost + replacement cost. Pregnancy per AI (P/AI) 38 ± 3 d after first AI was greatest for AIE heifers (61.1%) followed by CIDR5 (44.8%) and CIDR7 (35.7%) heifers. Furthermore, P/AI 73 ± 7 d after first AI was greatest for AIE (58.8%) and tended to be greater for CIDR5 (42%) than for CIDR7 (34.1%) heifers. The percentage of heifers that had spontaneous luteolysis from CIDR insertion to CIDR removal was reduced for CIDR5 compared with CIDR7 (13.8 vs 31.8%). Pregnancy rate was greatest for AIE heifers but did not differ between CIDR5 [adjusted hazard ratio (95% confidence interval)=0.75 (0.63, 0.90)] and CIDR7 [adjusted hazard ratio (95% confidence interval)=0.65 (0.54, 0.77)] heifers. Consequently, rearing cost and input cost of AIE heifers ($67.1 ± 4.4 and -$107.1 ± 7.0, respectively) were reduced compared with CIDR5 ($86.9 ± 5.1 and -$143.4 ± 8.1, respectively) and CIDR7 ($98.3 ± 5.1 and -$156.5 ± 8.2, respectively) heifers, but no differences were observed between CIDR5 and CIDR7 heifers.

Short communication: characterization of early postpartum estrous behavior and ovulation in lactating dairy cows using radiotelemetry.

The objective of this study was to describe early postpartum estrous behavior and ovulation in lactating dairy cows using radiotelemetry. Cows (n=50) were continuously monitored for behavioral estrus with a radiotelemetric system, HeatWatch II (CowChips LLC, Manalapan, NJ), from d 14 to approximately d 49 postpartum. Blood collection for analysis of progesterone and ovarian ultrasonography were performed once weekly starting on d 14. First ovulation was associated with behavioral estrus in 5 cows and occurred at 28.2±10.8 d (mean±SD; range 17 to 40 d). The average duration of estrus was 6.0±4.9 h (range 3 to 12.2 h), and the mean number of standing events was 18.4±8.9 (range 4 to 26). Based on progesterone concentrations of ≥1 ng/mL, estimated first postpartum ovulation occurred at 25.1±10.4 d (range 10 to 49 d) for 38 animals without evidence of behavioral estrus. The interval to estimated first ovulation without behavioral estrus was not different from the interval to first ovulation associated with behavioral estrus. Level of milk production and body condition score loss did not affect the interval to estimated first ovulation without estrus or first ovulation associated with estrus. Six animals did not show evidence of ovulation based on progesterone concentration, whereas 1 cow showed evidence of estrous behavior on the day before removal from the study. The majority of first postpartum ovulations (38/43; 88.4%) were not associated with behavioral estrus.

Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AI of Nelore cows and heifers.

The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N = 3; FAA-positive: N = 3) and the ability to produce neat semen with ≥ 70% morphologically normal sperm and 60% estimated progressive motility before cryopreservation. In Experiment 1, suckled multiparous Nelore cows (N = 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). On Day 9 the CIDR was removed, 12.5 mg of PGF(2α) and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day 11 (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P < 0.05) and BCS tended to affect P/TAI (P = 0.09), as cows with BCS ≥ 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS < 2.75. In Experiment 2, nulliparous Nelore heifers (N = 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. On Day 7, all heifers received PGF(2α) (12.5 mg). On Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P = 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI.

Effect of clinical mastitis and other diseases on reproductive performance of Holstein cows.

The objective of this study was to evaluate the effect of clinical mastitis and (or) other diseases on reproductive performance in lactating Holstein cows. Cows (n=967) from a commercial dairy farm were divided into four groups retrospectively: cows with clinical mastitis and other diseases (MD, n=54), clinical mastitis only (M, n=154), other diseases only (D, n=187), and cows with no record of clinical mastitis or other diseases (H, n=572). Days in milk at first service (DIMFS), services per conception (S/C), days not pregnant (DNP), the rate at which animals became pregnant over time and the proportion of cows that remained non-pregnant during 224 days of lactation were evaluated. Groups MD and M had greater (P<0.05) DNP compared with H (155+/-15 and 140+/-5 vs. 88+/-2, respectively). Moreover, MD and M had greater (P<0.05) S/C compared with H (3.0+/-0.4 and 2.1+/-0.1 vs. 1.6+/-0.1, respectively). The rate at which animals became pregnant over time was less (P<0.05) for MD and M and tended (P=0.1) to be less for D when compared with H. In addition, proportion of cows that remained non-pregnant by 224 days of lactation was greater (P<0.05) in MD, M, and D compared with H. Cows with mastitis were also divided into three groups according to the day of occurrence of the first case of clinical mastitis: (1) clinical mastitis occurred before 56 days postpartum (MP1); (2) clinical mastitis occurred between 56 and 105 days after parturition (MP2); and (3) clinical mastitis occurred after 105 days postpartum (MP3) Regardless of the time of occurrence, DNP was greater (P<0.05) for cows with mastitis compared with H. Time of mastitis occurrence affected S/C in that cows in MP2 and MP3 had a greater S/C compared with H cows (P<0.05). Reproductive efficiency was decreased by the presence of clinical mastitis alone because a greater proportion of cows with mastitis remained non-pregnant over time. Moreover, a greater proportion of cows with mastitis or diseases remained non-pregnant by 224 postpartum. Furthermore, the negative effects on reproduction were exacerbated when cows experienced both clinical mastitis and other diseases.

Effect of breeding protocols and reproductive tract score on reproductive performance of dairy heifers and economic outcome of breeding programs.

The objectives of this study were to evaluate the effect of reproductive protocols and reproductive tract score on reproductive performance of dairy heifers and economic outcomes of breeding programs. Holstein heifers (n = 534), 13 +/- 1 mo of age, were randomly assigned to 1 of 4 reproductive protocols. On the day of enrollment (d 0), heifers were palpated per rectum and received a score according to the maturity of their reproductive tract (1 = prepubertal; 2 = peripubertal; and 3 = puber-tal). Estrous detection-control heifers (CON, n = 146) received no treatment and were inseminated on detection of estrus for 28 d. Prostaglandin F(2alpha)-treated heifers (PGED, n = 137) received 1 injection of PGF(2alpha) on d 0 and were inseminated on detection of estrus; heifers not in-seminated by d 14 received a second injection of PGF(2alpha) and were observed for estrus and artificial insemination (AI) for an additional 14 d. Heifers enrolled in the estrous detection-timed AI (EDTAI, n = 140) treatment received a controlled internal drug-release (CIDR) insert on d 0, and 7 d later, the CIDR was removed and all heifers received an injection of PGF(2alpha), heifers received AI on detection of estrus, and those not inseminated by 72 h after PGF(2alpha) received an injection of GnRH concurrent with AI. Heifers in the GnRH-timed AI (GTAI, n = 111) treatment received 1 injection of GnRH on d 0, on d 6 heifers received a CIDR insert and injections of GnRH and PGF(2alpha), on d 13 the CIDR was removed and heifers received an injection of PGF(2alpha), and 48 h later all heifers received an injection of GnRH and AI. Pregnancy was diagnosed at 32 +/- 3 and 62 +/- 3 d after AI. Cost of reproductive protocols and their economic outcomes were calculated for a 28 d period beginning at enrollment. Heifers in the PGED treatment were inseminated at a faster rate than CON heifers. A smaller proportion of prepubertal and peripubertal heifers were inseminated within 14 d of enrollment compared with pubertal heifers. Pregnancy per AI of CON and PGED heifers was greater compared with EDTAI and GTAI heifers. Proportion of GTAI heifers pregnant at the end of the 28-d breeding program was or tended to be smaller compared with PGED and CON heifers, respectively. Heifers in the CON and PGED treatments had the smallest cost per pregnancy followed by heifers in the EDTAI and GTAI treatments, respectively. When different scenarios were evaluated, however, the mean cost per pregnancy was smallest for PGED heifers. Cost per pregnancy generated was greatest for prepubertal heifers, whereas pubertal heifers had the smallest cost per pregnancy generated. Treatment of dairy heifers with PGF(2alpha) every 14 d until insemination and pregnancy results in the best economic outcomes, and screening heifers according to RTS may prove beneficial to identify heifers that may not be pubertal and would have compromised reproductive and economic performance in a breeding program.

Effect of synchronization protocols on follicular development and estradiol and progesterone concentrations of dairy heifers.

The objectives were to evaluate the effect of synchronization protocols on follicular development and estradiol 17-beta (E(2)) and progesterone (P(4)) concentrations in dairy heifers. In experiment 1, 36 heifers were assigned to 1 of 6 synchronization protocols in a 3 x 2 factorial design: presynchronization with GnRH on study d -6 or -9 [study d 0 = initiation of the Cosynch + CIDR (controlled internal drug releasing insert containing P(4)) protocol] or no presynchronization (control) and one injection of PGF(2 alpha) or not on study d 0. In experiment 2, 126 heifers were assigned to 1 of 4 synchronization protocols in a 2 x 2 factorial arrangement: presynchronization or not with GnRH on study d -6 and injection of PGF(2 alpha) or not on study d 0. In experiments 1 and 2, all heifers received a modified Cosynch protocol with CIDR for 7 d starting on study d 0. After the PGF(2 alpha) of the Cosynch and removal of the CIDR, heifers were detected in estrus and inseminated. Those not inseminated by study d 10 received an injection of GnRH and were timed-inseminated. Ovaries were scanned by ultrasound on d 0, 2, and 5, daily from d 7 to 14, and on d 16. Blood samples collected on d 0, 2, 7, 9, and 16 were analyzed for P(4), and the blood sample collected on d 9 was analyzed for E(2). Pregnancy was diagnosed at 28 and 40 +/- 3 d after artificial insemination. In experiment 1, there was a tendency for the presynchronization protocol to affect the proportion of heifers ovulating in response to the first GnRH injection of the Cosynch + CIDR protocol. In experiment 2, a greater proportion of presynchronized heifers ovulated in response to the first GnRH injection. Although heifers receiving PGF(2 alpha) had larger ovulatory follicles on d 7 and before ovulation and shorter intervals to estrus and ovulation, these heifers tended to have decreased concentrations of E(2) during proestrus. Presynchronization of dairy heifers with GnRH increased ovulation in response to the first GnRH injection, and treatment of heifers with PGF(2 alpha) at initiation of the Cosynch + CIDR protocol increased the size of the ovulatory follicle and reduced the intervals to estrus and ovulation.

Correlation between reproductive status and steady-state messenger ribonucleic acid levels of the Myxovirus resistance gene, MX2, in peripheral blood leukocytes of dairy heifers.

The objectives of the current study were to evaluate the correlation between reproductive status and steady-state levels of Myxovirus resistance gene (MX2) mRNA in peripheral blood leukocytes (PBL) of dairy heifers and the reliability of using change in MX2 messenger RNA (mRNA) for identification of nonpregnant heifers 18 to 19 d after AI. Holstein heifers (n = 266), 13 +/- 1 mo of age, were assigned randomly to be inseminated (BRED; n = 214) or not (NONBRED; n = 52). Estrous cycles of all heifers were synchronized with an intravaginal insert containing progesterone for 7 d. At insert removal, heifers received an injection of PGF2alpha. Heifers in the BRED group were inseminated on detection of estrus or at a fixed time, 72 h after insert removal concomitant with a GnRH treatment. Heifers in the NONBRED group received an injection of GnRH 48 h after insert removal. Blood samples collected on d 0 (d of AI or estrus) and 18 were used to determine steady-state levels of MX2 mRNA. Samples collected on d 0, 7, 14, and 21 were analyzed for progesterone concentration. Pregnancy was determined retrospectively by progesterone concentration on d 21 and was diagnosed at 30 +/- 1 and 60 +/- 3 d after AI. The fold change in levels of MX2 mRNA from d 0 to 18 was greater for heifers classified and diagnosed as pregnant on d 21 (P < 0.05) and 30 +/- 1 (P < 0.05) and 60 +/- 3 (P < 0.05) d after AI compared with nonpregnant (bred but not pregnant) and NONBRED heifers. Heifers that experienced pregnancy loss from 21 to 30 +/- 1 (P = 0.11) or 21 to 60 +/- 3 (P = 0.08) d of gestation tended to have smaller fold increases in MX2 mRNA expression than those that maintained pregnancy. The sensitivity (range 57.1 to 65.6%) and negative predictive values (range 47.9 to 57.1%) of determining reproductive status on d 18 according to the change in the level of MX2 mRNA expression in PBL were low, and the correlation between diagnosis of pregnancy by fold change in MX2 mRNA expression and other methods was small (r = 0.20 to 0.36). The current study indicates that increased expression of MX2 mRNA in PBL is related to pregnancy approximately 21, 30, and 60 d after AI in dairy heifers and that losses that occurred later in pregnancy were associated with lower fold increases in MX2 mRNA. However, using the change in MX2 mRNA expression was not a reliable method for diagnosis of pregnancy at 18 d after AI because of the low sensitivity and negative predictive value.

Time and incidence of ovulation and conception rates after incorporating estradiol cypionate into a timed artificial insemination protocol.

Two experiments were conducted to determine the effect of estradiol cypionate (ECP), when incorporated into a conventional GnRH-PGF(2alpha)-GnRH timed artificial insemination protocol (Ovsynch), on systemic estradiol (E(2)), time and incidence of ovulation, luteal development, and conception rate in Holstein cows. Our objective was to determine if administration of 0.25 mg of ECP at the time of the second GnRH injection would effectively synchronize ovulation and increase conception rate. In Experiment 1, lactating Holstein cows (n = 23; 58.7 +/- 1.2 d in milk) were synchronized with PGF(2alpha) (at d -10). Ten days later, Ovsynch was initiated with the administration of 100 mug of GnRH (d 0) followed by PGF(2alpha) on d 7. On d 9, cows were assigned randomly to be treated with either GnRH + 0.25 mg of ECP (OVS-ECP; n = 11) or GnRH and 1 mL of cottonseed oil (OVS-C; n = 12). Ovarian activity was monitored by ultrasonography on d 0, 7, and 9. To determine the time of ovulation, ultrasound examinations were conducted at 12 and 20 h posttreatment and then at least every 3 h until either 36 h posttreatment or ovulation was observed. Blood samples were collected on d 0, 7, 9, and 16 for progesterone analysis. Blood samples also were collected at the time of treatment (d 9, 0 h) and at 6, 12, 20, and 28 h for E(2) analysis. Incidence of ovulation did not differ between treatments. Mean ovulation time relative to the second GnRH administration was similar between treatments. Serum progesterone concentration did not differ between treatments at any time. Serum E(2) concentration was not different at the time of treatment (0 h); however, mean E(2) concentration was greater for the OVS-ECP group at 6 and 12 h after treatment compared with OVS-C. In Experiment 2, lactating dairy cows (n = 333) in 3 commercial herds were randomly assigned to OVS-ECP (n = 169) or OVS-C (n = 164). Cows were inseminated 22 to 24 h posttreatment. Conception rates did not differ between treatments. Estradiol cypionate treatment was successful in increasing serum E(2) when administered at the time of the second dose of GnRH in the Ovsynch protocol. Conception rates, however, were not affected by treatment.

Heterozygosity for a protein truncation mutation of sodium channel SCN8A in a patient with cerebellar atrophy, ataxia, and mental retardation.

BACKGROUND: The SCN8A gene on chromosome 12q13 encodes the voltage gated sodium channel Na(v)1.6, which is widely expressed in neurons of the CNS and PNS. Mutations in the mouse ortholog of SCN8A result in ataxia and other movement disorders. METHODS: We screened the 26 coding exons of SCN8A in 151 patients with inherited or sporadic ataxia. RESULTS: A 2 bp deletion in exon 24 was identified in a 9 year old boy with mental retardation, pancerebellar atrophy, and ataxia. This mutation, Pro1719ArgfsX6, introduces a translation termination codon into the pore loop of domain 4, resulting in removal of the C-terminal cytoplasmic domain and predicted loss of channel function. Three additional heterozygotes in the family exhibit milder cognitive and behavioural deficits including attention deficit hyperactivity disorder (ADHD). No additional occurrences of this mutation were observed in 625 unrelated DNA samples (1250 chromosomes). CONCLUSIONS: The phenotypes of the heterozygous individuals suggest that mutations in SCN8A may result in motor and cognitive deficits of variable expressivity, but the study was limited by lack of segregation in the small pedigree and incomplete information about family members. Identification of additional families will be required to confirm the contribution of the SCN8A mutation to the clinical features in ataxia, cognition and behaviour disorders.

Conception rates and serum progesterone concentration in dairy cattle administered gonadotropin releasing hormone 5 days after artificial insemination.

The objectives of this study were to determine the effect of administration of exogenous GnRH 5days after artificial insemination (AI) on ovarian structures, serum progesterone concentration, and conception rates in lactating dairy cows. In experiment 1, 23 Holstein cows were synchronized using the Ovsynch protocol. Five days after AI (day 0) cows were assigned randomly to receive either saline (saline; n=11) or 100microg GnRH (GnRH; n=12). To examine ovarian structures, ultrasonography was performed on day 1 and every other day beginning on day 5 until day 13. On days 5 and 13 blood samples were obtained to measure serum progesterone concentrations. All cows in the GnRH-treated group developed an accessory corpus luteum (CL), whereas cows in the saline group did not. Mean serum progesterone concentrations did not differ between GnRH and saline groups on day 5 (1.64+/-0.46ng/ml versus 2.04+/-0.48ng/ml). On day 13 serum progesterone concentrations were greater (P<0.05) in the GnRH group compared with saline (5.22+/-0.46ng/ml versus 3.36+/-0.48ng/ml). In experiment 2, 542 lactating cows, at two different commercial dairies, were used to test the effect of administering GnRH 5 days after AI on conception rates. Cows were synchronized and detected for estrus according to tail chalk removal. Cows detected in estrus received AI within 1h after detection of estrus. Five days after AI, cows were assigned randomly to receive either GnRH (n=266) or saline (n=276). Pregnancy status was determined by palpation per rectum of uterine contents approximately 40 days after AI. There was no effect of farm on conception rate. There was no effect of treatment as conception rates did not differ between GnRH and saline groups (26.7% GnRH versus 24.3% saline). Regardless of treatment, days in milk, parity, milk yield, and number of services had no effect on the odds ratio of pregnancy. In summary, the results of this study indicated that GnRH administered 5 days after AI increased serum progesterone by developing an accessory CL but did not improve conception rates in dairy cattle.

Short communication: Conception rates following detection of estrus and timed AI in dairy cows synchronized using GnRH and PGF2alpha.

The objective of this study was to compare conception rates of cows exhibiting spontaneous estrus and receiving artificial insemination (AI) before completion of a timed AI protocol with cows that did not display estrus spontaneously, but were inseminated after 1 of 3 GnRH-PGF2alpha protocols. Cows (n = 432) in 2 herds were administered GnRH on d -7 and were tail-chalked daily. Cows detected in estrus before d 0 were inseminated immediately. Cows not detected in estrus by d 0 were administered PGF2alpha and were tail-chalked daily until 48 h after PGF2alpha. Cows detected in estrus from d -7 to 48 h after PGF2alpha were inseminated and designated as treatment A (n = 46). Cows not detected in estrus and not inseminated by 48 h after PGF2alpha were assigned randomly to receive either GnRH 48 h after PGF2alpha and timed AI 16 h later (treatment B; n = 132), or GnRH and timed AI 64 h after PGF2alpha (treatment C; n = 127), or timed AI 64 h after PGF2alpha (treatment D; n = 127). Pregnancy was diagnosed 38 to 45 d after AI by palpation per rectum of uterine contents. Nearly 11% of all cattle exhibited spontaneous estrus and received immediate AI. Herd did not influence the percentage of cows detected in estrus and inseminated. Conception rates did not differ among treatments. Conception rates differed between herds, but no interaction of herd x treatment was detected. No differences were detected between herds for days in milk, milk production, AI service number, or parity.

Effect of simultaneous thawing of multiple 0.5-mL straws of semen and sequence of insemination on conception rate in dairy cattle.

The objectives of this study were to determine 1) the effect of simultaneous thawing of multiple 0.5-mL straws of semen and sequence of insemination (first, second, third, or fourth) on conception rates in dairy cattle, 2) whether the conception rates achieved following AI by professional AI (PAI) technicians and herdsman-inseminators (HI) differed, and 3) the effect of elapsed time from initiation of thawing straws of semen to seminal deposition on conception rates in dairy cattle. Four dairies with PAI and four with HI participated in the study. Initial data recorded included beginning thaw time, cow identification number, and time of seminal deposition. Herd records were retrieved following pregnancy diagnosis. Conception rates of dairy cows (n = 1025) were not affected by sequence of insemination (first, second, third, or fourth). Conception rates for herds using PAI were 40, 47, 41, and 50%. Conception rates for herds using HI were 24, 20, 33, and 30%. Average conception rates of dairy cows differed between PAI and HI (45 vs. 27%, respectively). The difference in mean conception rate achieved by PAI and HI was not attributable to milk production, parity, service number or stage of lactation. The elapsed time from initial thaw to completion of fourth AI was shorter for PAI than for HI, 7.6 vs. 10.9 min, respectively. Although the average conception rate differed between PAI and HI, elapsed time from initial thaw to completion of fourth AI and sequence of insemination (first, second, third, or fourth) had no effect on conception rate within inseminator group.

Insulin receptor splicing alteration in myotonic dystrophy type 2.

Myotonic dystrophy (DM) is caused by either an untranslated CTG expansion in the 3' untranslated region of the DMPK gene on chromosome 19 (dystrophia myotonica type 1 [DM1]), or an untranslated CCTG tetranucleotide repeat expansion in intron 1 of the ZNF9 gene on chromosome 3 (dystrophia myotonica type 2 [DM2]). RNA-binding proteins adhere to transcripts of the repeat expansions that accumulate in the nucleus, and a trans-dominant dysregulation of pre-mRNA alternative splicing has been demonstrated for several genes. In muscle from patients with DM1, altered insulin-receptor splicing to the nonmuscle isoform corresponds to the insulin insensitivity and diabetes that are part of the DM phenotype; because of insulin-receptor species differences, this effect is not seen in mouse models of the disease. We now demonstrate that comparable splicing abnormalities occur in DM2 muscle prior to the development of muscle histopathology, thus demonstrating an early pathogenic effect of RNA expansions.

Molecular genetics of spinocerebellar ataxia type 8 (SCA8).

We previously reported that a transcribed but untranslated CTG expansion causes a novel form of ataxia, spinocerebellar ataxia type 8 (SCA8) (Koob et al., 1999). SCA8 was the first example of a dominant spinocerebellar ataxia that is not caused by the expansion of a CAG repeat translated into a polyglutamine tract. This slowly progressive form of ataxia is characterized by dramatic repeat instability and a high degree of reduced penetrance. The clinical and genetic features of the disease are discussed below.

Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum.

BACKGROUND: Myotonic dystrophy types 1 (DM1) and 2 (DM2/proximal myotonic myopathy PROMM) are dominantly inherited disorders with unusual multisystemic clinical features. The authors have characterized the clinical and molecular features of DM2/PROMM, which is caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. METHODS: Three-hundred and seventy-nine individuals from 133 DM2/PROMM families were evaluated genetically, and in 234 individuals clinical and molecular features were compared. RESULTS: Among affected individuals 90% had electrical myotonia, 82% weakness, 61% cataracts, 23% diabetes, and 19% cardiac involvement. Because of the repeat tract's unprecedented size (mean approximately 5,000 CCTGs) and somatic instability, expansions were detectable by Southern analysis in only 80% of known carriers. The authors developed a repeat assay that increased the molecular detection rate to 99%. Only 30% of the positive samples had single sizeable expansions by Southern analysis, and 70% showed multiple bands or smears. Among the 101 individuals with single expansions, repeat size did not correlate with age at disease onset. Affected offspring had markedly shorter expansions than their affected parents, with a mean size difference of -17 kb (-4,250 CCTGs). CONCLUSIONS: DM2 is present in a large number of families of northern European ancestry. Clinically, DM2 resembles adult-onset DM1, with myotonia, muscular dystrophy, cataracts, diabetes, testicular failure, hypogammaglobulinemia, and cardiac conduction defects. An important distinction is the lack of a congenital form of DM2. The clinical and molecular parallels between DM1 and DM2 indicate that the multisystemic features common to both diseases are caused by CUG or CCUG expansions expressed at the RNA level.

Effect of time of insemination on number of accessory sperm, fertilization rate, and embryo quality in nonlactating dairy cattle.

Two experiments were conducted to determine the effect of insemination time on number of accessory sperm per embryo (ovum), fertilization rate, and embryo quality. Semen was collected from three fertile Holstein bulls and cryopreserved in egg yolk-citrate-glycerol. In experiment 1, cows were continuously monitored for behavioral estrus by the HeatWatch estrous detection system and were artificially inseminated (AI) with one 0.5-ml straw (25 x 10(6) sperm) at the onset of estrus (AI 0 h), 12 h after onset (AI 12 h), or received natural service at 0 h (Nat 0 h) from one of three bulls. From 150 inseminations, 115 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; Nat 0 h: n = 37) were recovered 6 or 7 d after insemination. Fertilization rates differed between treatments (AI 0 h: 67%; AI 12 h: 79%; Nat 0 h: 98%). Median accessory sperm per embryo (ovum) also differed (AI 0 h: 1; AI 12 h: 10; and Nat 0 h: 27) and paralleled the fertilization rate. Embryo quality was not affected by insemination time or natural service. In experiment 2, cows received AI at 0, 12, or 24 h (AI 24 h) after the onset of estrus as determined by HeatWatch. From 154 inseminations, 117 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; AI 24 h: n = 39) were recovered 6 or 7 d after insemination. Fertilization rates did not differ in experiment 2 (AI 0 h: 66%; AI 12 h: 74%; AI 24 h: 82%); however, a trend toward a higher fertilization rate accompanied AI 24 h. Median accessory sperm values increased from AI 0 h (1) to AI 24 h (4). Embryo quality declined with AI at increasing intervals after onset of estrus, as percentages of excellent and good, fair and poor, and degenerate embryos were as follows: 77, 15, 8; 52, 38, 10; and 47, 19, 34 for the 0-, 12-, and 24-h inseminations, respectively. Results indicate AI 12 h after the onset of estrus provides a compromise between potential fertilization failure (AI 0 h) and embryo failure (AI 24 h), despite increased accessory sperm per embryo (ovum) after AI 24 h. Artificial insemination 12 h after onset of estrus should optimize fertility of dairy cattle through an acceptable fertilization rate, number of accessory sperm per embryo, and desirable embryo quality.