Effect of housing type on health and performance of preweaned dairy calves during summer in Florida.

The objective was to evaluate the effect of housing type on health and performance of preweaned dairy calves. One hundred calves, Holsteins (HO) or Holstein-Jersey crosses (HJ), were randomly assigned to a Calf-Tel (L. T. Hampel Corp., Germantown, WI) polyethylene hutch exposed to direct sun light (n=25 for both HO and HJ) or a wire hutch with a plywood shade located under trees (n=24 and 26 for HO and HJ, respectively). Calf rectal temperature, respiratory rate, health scores, and weaning weight were compared by housing type. Rectal temperatures were higher in calves in Calf-Tel hutches compared with calves in wire hutches at 1500 h, at 40.1±0.28 and 39.1±0.22°C, respectively. Similarly, respiratory rates were higher in calves in Calf-Tel hutches compared with calves in wire hutches at 1500 h, at 90±15 and 65±10 breaths per minute, respectively. Frequencies of calves presenting abnormal ear scores did not differ between calves in Calf-Tel or wire hutches. Abnormal eye scores were less likely to occur for calves in the wire hutches. Frequencies of calves with signs of respiratory disease, such as nasal discharge and coughing, were higher in the wire hutches compared with the Calf-Tel hutches. No differences were noted in the incidence and time of first diarrhea event in calves between hutch types. Calves in wire hutches had 5.47 greater odds of receiving veterinary treatment compared with calves in the Calf-Tel hutches. Weaning weights were not different for calves in Calf-Tel or in wire hutches. The lower frequency of calves with abnormal health scores and receiving veterinary treatment in Calf-Tel hutches and the lack of difference in weight gain suggests that this housing system adapted well to the specific environmental conditions of this study.

Application of one injection of prostaglandin F(2alpha) in the five-day Co-Synch+CIDR protocol for estrous synchronization and resynchronization of dairy heifers.

The objective was to determine if the 5-d Co-Synch+CIDR (controlled internal drug releasing insert) protocol can be used in dairy heifers for a synchronized timed artificial insemination (TAI) with one injection of PGF(2alpha) for first and second services. In experiment 1, heifers were assigned randomly to receive 1 (n=295) or 2 (n=298) injections of PGF(2alpha) in the 5-d Co-Synch+CIDR protocol. Corpus luteum (CL) regression was measured in one replicate (n=218). No difference in pregnancy per TAI (P/TAI; 46.1 and 48.6%) or CL regression (86.9 and 92.8%) was detected for 1 versus 2 injections of PGF(2alpha), respectively. In experiment 2, nonpregnant heifers (n=86) were assigned to a resynchronized 5-d Co-Synch+CIDR with 1 PGF(2alpha)/TAI or insemination at detected estrus. There was no difference in P/TAI (52.2 and 55%) between groups. In experiment 3, nonpregnant heifers (n=110) were assigned randomly to receive a CIDR (n=54) or no CIDR insert (n=56) in the 5-d Co-Synch protocol for resynchronization of TAI. Pregnancy per TAI was lower without the CIDR device (39.3 vs. 51.8%). In a commercial field evaluation, 416 heifers were synchronized for the first and resynchronized TAI with the 5-d Co-Synch+CIDR protocol with 1 injection of PGF(2alpha). Pregnancy per TAI on d 60 was 58.2 and 47.5% for first and second TAI, respectively; there was a sire effect to the second TAI. In conclusion, the 5-d Co-Synch+CIDR protocol with 1 injection of PGF(2alpha) is an effective reproductive management program for first and second TAI in dairy heifers.

Comparison of reproductive performance in lactating dairy cows bred by natural service or timed artificial insemination.

The objective of this study was to compare reproductive performance of lactating dairy cows bred by natural service (NS) or timed AI (TAI). One thousand fifty-five cows were blocked by parity and enrolled to receive either NS or TAI. Cows in both groups were presynchronized with 2 injections of PGF(2alpha) given at 42 and 56 d postpartum. Fourteen days after the last PGF(2alpha) injection, cows in the TAI group were enrolled in an Ovsynch protocol (d 0 GnRH; 7 d later, PGF(2alpha); 56 h after PGF(2alpha) injection, second dose of GnRH; and 16 h after second GnRH cows were TAI). Cows in the TAI group were resynchronized with an intravaginal insert containing progesterone inserted 18 d after TAI and removed 7 d later when GnRH was given. Cows were examined by ultrasonography on d 32 after TAI; nonpregnant cows received PGF(2alpha) and GnRH 56 h later followed by TAI 16 h after the GnRH injection. Nonpregnant cows in TAI group were reinseminated up to 5 times using the same scheme. Cows in the NS group were exposed to bulls 14 d after the second PGF(2alpha) injection, and ultrasonography was performed 42 d after exposure to bulls to determine pregnancy status. Nonpregnant cows in the NS group were reexamined by transrectal palpation combined with ultrasound every 28 d until diagnosed pregnant or 223 d postpartum, whichever occurred first. Cows diagnosed pregnant in TAI or NS were reconfirmed 28 d later to determine pregnancy loss. All bulls underwent an evaluation of breeding soundness and were rested for 14 d after 14 d of cow exposure. Health disorders were evaluated up to 70 d postpartum, and body condition score was evaluated at d 70 postpartum. Blood was collected on d 56 and 65 postpartum and analyzed for progesterone to determine cyclicity. The proportion of pregnant cows in the first 21 d of breeding did not differ between groups. The overall 21-d cycle pregnancy rate (PR), which included a total of 8 and 5 service opportunities for NS and TAI, respectively, was not different between groups (25.7 and 25.0% for NS and TAI, respectively). The daily rate of pregnancy was 15% greater for NS than TAI because cows in NS had a greater PR, which resulted in fewer median days open (111 vs. 116 d). Proportion of pregnant cows at 223 d postpartum was greater in the NS than TAI group (84.2 vs. 74.8%, respectively). Cyclicity did not affect reproductive performance. Cows with body condition score >/=2.75 had greater proportion of pregnant cows in the first 21 d of breeding and daily PR in the first 223 d postpartum Primiparous cows had greater proportion of pregnant cows and daily PR than multiparous cows at 223 d postpartum. In conclusion, the greater proportion of pregnant cows in the NS group was attributed to more opportunities for breeding than in the TAI group.

Rectal temperature, calving-related factors, and the incidence of puerperal metritis in postpartum dairy cows.

The objectives of this study were as follows: 1) to evaluate the association among abnormal calving, parity, and season on the incidence of puerperal metritis (PM) and clinical endometritis (CE) during d 3 to 13 and 20 to 30 postpartum, respectively; 2) to describe the rectal temperature (RT) of cows with PM before diagnosis; and 3) to document associations among PM, CE, and reproductive performance in lactating dairy cows. This study followed a prospective observational study design. Cows were classified as having an abnormal calving status (AC), i.e., cows calving with dystocia, twins, retained fetal membranes, or some combination of these conditions, and having a normal calving status (NC). Daily RT was recorded from d 3 to 13 postpartum for all cows, and health examinations were performed on cows that appeared not well. A total of 450 calvings were evaluated. Cows with an AC had greater odds of PM than cows with NC [adjusted odds ratio (AOR) = 4.8; 95% confidence interval (CI) = 2.9 to 8.0). A season by parity interaction showed that primiparous cows that calved during the warm season had lower AOR of PM than during the cool season (0.24; 95% CI = 0.09 to 0.62), whereas multiparous cows did not have seasonal effects on PM (1.43; 95% CI = 0.65 to 3.18). Cows with AC have greater AOR for CE than cows with NC (2.8; 95% CI = 1.7 to 4.9), and greater AOR of CE were detected in cows diagnosed with PM than in cows without PM (2.2; 95% CI = 1.1 to 3.9). Rectal temperature in cows with PM increased significantly 24 h before diagnosis of PM, reaching 39.2 +/- 0.05 degrees C on the day of diagnosis. In cows with PM and fever at diagnosis, the RT began to increase from 72 to 48 h before the diagnosis of PM and continued to increase to 39.7 +/- 0.09 degrees C on d 0 (day of diagnosis). Nonetheless, cows with PM without fever at diagnosis had no daily increases in RT before diagnosis of PM. Still, the RT on d 0 was different from cows without PM. Cows without PM had a stable RT (38.6 +/- 0.01 degrees C). There were no detected differences in first-service conception risk or cumulative pregnancy risk by 150 d postpartum between cows with or without PM. Still, a season effect on first-service conception AOR (warm vs. cool = 0.98; 95% CI = 0.18 to 0.72) and accumulated pregnancy AOR by 150 d postpartum was detected (warm vs. cool = 0.18; 95% CI = 0.10 to 0.33).

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress.

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.

Fetal survival in the cow after pregnancy diagnosis by palpation per rectum.

It is desirable to determine whether a cow has failed to become pregnant as early as possible, preferably prior to 50 d after insemination. Although palpation per rectum has been the generally accepted method of pregnancy diagnosis in cattle, the procedure may be a significant iatrogenic cause of fetal attrition. In a study conducted at a North Florida dairy from January through June 1982, pregnancy was determined in 192 Holstein-Friesian cows by measuring low milk progesterone (P4) content on day of insemination (Day 0) and elevated P4 on Days 21 and 24. Pregnant cows were randomly assigned to a treatment and a control group. Cows in the treatment group (n=85) were palpated per rectum twice between Days 42 and 46 after insemination. Cows in the control group (n=107) were not palpated until both groups were palpated at Day 90. Palpation, done by two experienced clinicians, consisted of palpation of fetal fluid fluctuation, identification of the amniotic vesicle, and slipping of the chorioallantoic membranes. In both groups of cows fetal viability was monitored by milk P4 content. Last milk (5 to 15 ml) was collected from one front quarter on Days 0, 21, and 24 and twice weekly thereafter through Day 63. Milk was defatted by centrifugation and the fat-free milk progesterone content measured by a radioimmunoassay without extraction. The milk P4 test was 80.0% accurate in determining pregnancy in the palpated and nonpalpated cows. In the cows palpated on Days 42 to 46, pregnancy rates declined by 7.5% as determined by palpation at Day 90, or by 11.4% as determined by milk P4 content through Day 63 (both values P<0.05). Cows that were not palpated on Days 42 to 46 showed a 1.9% increase or a 4.3% decline in pregnancy rates as determined by the same criteria. Before palpation, at Days 42 to 46, pregnancy rates were better in cows that were inseminated in winter (January to March) than in spring (April to June) (82.3% vs 61.6%; P<0.05); P4 content was higher (winter>spring=2.13 ng/ml vs 1.38 ng/ml; P<0.05). First-lactation cows had higher P4 values on Days 21 and 24 than older cows (P<0.01).

Breed and other effects on reproductive traits and breeding soundness categorization in young beef bulls in Florida.

Yearling, grass-fed, beef bulls at the USDA Subtropical Agricultural Research Station, Brooksville, Florida, were assessed for physical and semen traits in January, April, July and October of 1991 (Trial 1) and 1992 (Trial 2). Bulls were given a breeding soundness evaluation (BSE) using revised semen and scrotal circumference (SC) criteria. In Trial 1, the bulls consisted of Angus (n = 15), Brahman (n = 14), Hereford (n = 15) and Senepol (n = 14). In Trial 2, the breeds were Angus (n = 15), Brahman (n = 16), Romosinuano (n = 13) and Nellore x Brahman (n = 9). Trial bulls generally showed delayed growth compared with grain-fed bulls in temperate environments. Breed influenced semen traits (percentage sperm motility, normal spermatozoa and those with primary abnormalities) in both trials. Temperate Bos taurus breeds (Angus, Hereford) were generally superior to Bos indicus breeds (Brahman, Nellore x Brahman). Tropically-adapted Bos taurus breeds (Senepol, Romosinuano) were intermediate for those traits tested. In general, tropically-adapted Bos taurus breeds were more similar in reproductive development to temperate Bos taurus than to Bos indicus breeds. Breed by test period interactions occurred and were mainly influenced by delayed sexual maturity of Bos indicus bulls. Qualitative semen traits increased with bull age, particularly from 12 to 18 mo. Scrotal circumference development was slower in the Bos indicus breeds. Bulls of satisfactory BSE status at 18.1 to 22 mo of age were 73.9% in Trial 1 and 58.5% in Trial 2. Brahman bulls had the least satisfactory BSE scores in both years (Trial 1, 44.4%; Trial 2, 22.2%). Most bulls failed to achieve satisfactory BSE status due to a small SC relative to age (Trial 1, 66%; Trial 2, 72%). The most efficacious use of the BSE was > or = 15 mo in Bos taurus bulls and > 18 mo for Bos indicus bulls. Although the BSE has proven to be useful for the assessment of young, pasture-raised bulls in semi-tropical environments, use of SC thresholds linked more with growth traits than with calendar age would improve comparisons of relative reproductive development in such bulls, particularly those of Bos indicus derivation.

Cloned ovarian cells from sheep: Cholesterol dependency in progesterone synthesis.

The TV4 cell line is derived from sheep ovarian tissues trypsinized for 60 min and developed from a clone after serial dilutions. The TV4 cells had a doubling time of 24 h in B2 medium with 10% fetal calf serum and 10% BSA. TV4 cells synthesized progesterone (P4) in the presence of cholesterol. As the concentration of cholesterol increased (0, 92.5 and 125 mg/l), synthesis of P4 increased (P<0.01) from 1.05 +/- 0.20 to 30.6 +/- 3.03 ng/ml. Kinetics of P4 production were determined; a linear production response (y = 5.816 + 1.05 x, y = ng/ml, x = hour of incubation; R(2) = 0.97) was observed with up to 35 ng/ml of P4 obtained by 30 h of incubation. Follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, or FSH and testosterone did not have any effect on estradiol-17beta (E2) or P4 production. Aromatase activity measured by RIA and HPLC following incubation with either nonradiolabeled or labeled testosterone was undetectable. In conclusion, this study established a cell line from the sheep ovary which has a high ability of divide and produce progesterone.

The dominant follicle exerts an interovarian inhibition on FSH-induced follicular development.

An experiment was conducted to evaluate the role of the dominant follicle (DF) of the first wave in regulating follicular and ovulatory responses and embryonic yield to a superovulation regime with FSH-P. Twenty normally cycling Holstein-Freisian heifers (n = 20) were synchronized with GnRH and pgf(2alpha) and randomly assigned to a control or a treated group (n = 10 each). Treated heifers had the first wave dominant follicle removed via transvaginal, ultrasound-guided aspiration on Day 6 after a synchronized estrus. All heifers received a total of 32 mg FSH-P given in decreasing doses at 12 h intervals from Day 8 to Day 11 plus two injections of pgf(2alpha) (35 mg and 20 mg, respectively) on Day 10. Heifers were inseminated at 6 h and 16 h after onset of estrus. Follicular dynamics were examined daily by transrectal ultrasonography from Day 4 to estrus, once following ovulation, and at the time of embryo collection on Day 7. Blood samples were collected daily during the superovulatory treatment and at embryo collection. Follicles were classified as: small, /= 10 mm. Aspiration of the dominant follicle was associated with an immediate decrease in large follicles, and a linear rate increase in small follicles from Day 4 to Day 8 just prior to the FSH-P injections, (treatment > control: +0.33 vs. -0.22, number of small follicles per day; P < 0.10). During FSH-P injections, the increase in number of medium follicles was greater (P < 0.01) for treatment on Day 9-11 (treatment > control: Day 9, 3.2 > 1.8; Day 10, 9.2 > 4.7; Day 11, 13.1 > 8.3; +/- 0.56). Number of large follicles was greater in treatment at Day 11 (5.12 > 1.4 +/-0.21; P < 0.01). Mean number of induced ovulatory follicles (difference between number of follicles at estrus and Day 2 after estrus) was greater in treatment (13.4 > 6.3 +/- 1.82; P < 0.01). Plasma estradiol at Day 11 during FSH-P treatment was greater in treatment (32.5 > 15.8 +/- 2.6; P < 0.01). Plasma progesterone at embryo flushing (Day 7 after ovulation) was greater in treatment (7.4 > 4.9; P < 0.02); technical difficulties at embryo recovery reduced sensitivity of embryonic measurements. No changes in the distribution of unfertilized oocytes and embryo developmental stages were detected between control and treatment groups. Presence of dominant follicle of the first wave inhibited intraovarian follicular responses to exogenous FSH.

Effects of calving-related disorders on prostaglandin, calcium, ovarian activity and uterine involution in postrartum dairy cows.

Postpartum ovarian activity, uterine involution and plasma concentrations of calcium and 15-keto-13, 14 dihydro-prostaglandin F2alpha (PGFM) were assessed in dairy cows with retained fetal membranes (n=10) and milk fever (n=10) at parturition. In addition, calcium and PGFM were evaluated in dairy cows affected with uterine prolapse (n=10) and pyometra (n=14). Cows with retained fetal membrane averaged 24.2+/-3.7 d until their first postpartum ovulation, while controls averaged 29.0+/-3.7 d (P>0.10). In cows with retained fetal membranes, the difference in follicular activity between the contralateral and ipsilateral ovaries in relation to the previously gravid uterine horn was appreciably greater post partum when compared with that of the controls. Cows with milk fever had an average of 30.8+/-3.1 d until their first postpartum ovulation, while control cows had an average of 20.4+/-3.3 d (P<0.05). The mean diameter of the uterine horns in cows with milk fever was greater (P<0.05) compared with that of the controls between Days 15-32 post partum. Concentrations of plasma calcium were lower in cows with retained fetal membranes within 24 h after parturition and during the first week post partum than in the controls (6.27+/-0.18 vs 7.40+/-0.18 mg/100ml, P<0.05). Concentration of calcium was lower (P<0.05) in cows with milk fever on Day 1 prior to treatment (4.68+/-0.40 < 5.8+/-0.45 mg/100ml) than in control cows; however, the calcium (Ca) level was not different during the subsequent 7 d post partum after treatment. Cows with uterine prolapse had lower concentrations of Ca during the first 7 d post partum than the controls (6.10+/-0.15 vs 7.33+/-0.12mg/100ml; P<0.01). Cows with pyometra had higher (P<0.05) concentrations of plasma PGFM than the controls (208.+/-13.2 > 138.1+/-15.2).

Effects of buserelin injection and deslorelin (GnRH-agonist) implants on plasma progesterone, LH, accessory CL formation, follicle and corpus luteum dynamics in Holstein cows.

The influence of Buserelin injection and Deslorelin (a GnRH analogue) implants administered on Day 5 of the estrous cycle on plasma concentrations of LH and progesterone (P4), accessory CL formation, and follicle and CL dynamics was examined in nonlactating Holstein cows. On Day 5 (Day 1 = ovulation) following a synchronized estrus, 24 cows were assigned randomly (n = 4 per group) to receive 2 mL saline, i.m. (control), 8 micrograms, i.m. Buserelin or a subcutaneous Deslorelin (DES) implant in concentrations of 75 micrograms, 150 micrograms, 700 micrograms or 2100 micrograms. Blood samples were collected (for LH assay) at 30-min intervals for 2 h before and 12 h after GnRH-treatment from cows assigned to Buserelin, DES-700 micrograms and DES-2100 micrograms treatments and thereafter at 4-h intervals for 48 h. Beginning 24 h after treatment, ovaries were examined by ultrasound at 2-h intervals until ovulation was confirmed. Thereafter, ultrasonography and blood sampling (for P4 assay) was performed daily until a spontaneous ovulation before Day 45. A greater release of LH occurred in response to Deslorelin implants than to Buserelin injection (P < 0.01). Basal levels of LH between 12 and 48 h were higher in DES-700 micrograms group than in DES-2100 micrograms and Buserelin (P < 0.05). The first wave dominant follicle ovulated in all cows following GnRH treatment. Days to CL regression did not differ between treatments, but return to estrus was delayed (44.2 vs 27.2 d; P < 0.01) in cows of DES-2100 micrograms group. All GnRH treatments elevated plasma P4 concentrations, and the highest P4 responses were observed in the DES-700 micrograms and DES-2100 micrograms groups. The second follicular wave emerged earlier in GnRH-treated than in control cows (9.9 vs 12.8 d; P < 0.01). However, emergence of the third dominant follicle was delayed in cows of DES-2100 micrograms treatment (37.0 d) compared with DES-700 micrograms (22.2 d), Buserelin (17.8 d) or control (19.0 d). In conclusion, Deslorelin implants of 700 micrograms increased plasma P4 and LH concentrations and slightly delayed the emergence of the third dominant follicle. On the contrary, Deslorelin implants of 2100 micrograms drastically altered the P4 profiles and follicle dynamics.

Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida.

The objective was to compare conception rates to embryo transfer relative to AI, during summer heat stress, in lactating dairy cows. Holstein cows (n = 180; 50 to 120 d postpartum) were allocated randomly to 1 of 3 groups: artificial insemination (AI, n = 84), embryo transfer using either embryos collected from superovulated donors (ET-DON, n = 48), or embryos produced in vitro (ET-IVF, n = 48). Embryos from superovulated donors were frozen in 10% glycerol and were rehydrated in a 3-step procedure, in decreasing concentrations of glycerol in a sucrose medium before transfer. Embryos produced in vitro were frozen in 1.5 M ethylene glycol, thawed and transferred without rehydration. Blood samples were collected from AI and ET recipients on Days 0, 7 and 22 for measurement of progesterone in plasma. Conception rate was estimated for the three groups at Day 22 (progesterone > 1 ng/mL) and confirmed at Day 42 by palpation per rectum. Conception rate estimates at Day 22 did not differ among groups (AI, 60.7%; ET-DON, 60.4%; ET-IVF, 54.2%), but conception rates at Day 42 differed (AI, 21.4%; ET-DON, 35.4%; ET-IVF, 18.8%; AI versus ET: P > 0.10 and ET-DON versus ET-IVF: P < 0.05). In cows considered pregnant at 22 d but diagnosed open at 42 d, the interestrous intervals were 28.8 +/- 2.2, 35.2 +/- 3.5 and 31.6 +/- 2.9 d, respectively, for AI, ET-DON and ET-IVF groups. Transfer of embryos collected from nonheat-stressed superovulated donors significantly increased conception rates in heat stressed dairy cattle. However, transfer of IVF-derived embryos had no advantage over AI. Where appropriate mechanisms are in place to attenuate the effects of heat stress, embryo transfer using frozen-thawed donor embryos increases conception rates.

Dynamics of prostaglandin secretion, intrauterine fluid and uterine clearance in reproductively normal mares and mares with delayed uterine clearance.

Two experiments were performed to investigate relationships between oxytocin, prostaglandin release, uterine emptying and fluid accumulation in the uterus. In Experiment 1, the effect of oxytocin on the pattern of prostaglandin release during uterine clearance of radiocolloid was measured in 5 normal mares and 5 mares with delayed uterine clearance. Uterine clearance was measured during estrus by scintigraphy at 0, 60 and 120 min after colloid infusion. After the 120-min reading, 20 IU, i.v., oxytocin were given, and the amount of colloid cleared was measured at 135, 150 and 180 min. Plasma was obtained prior to and during scintigraphy at 5- and 15-min intervals to measure concentrations of 15-keto-13,14-dihydro-PGF2 alpha metabolite (PGFM) by RIA. In Experiment 2, plasma PGFM levels were compared after administration of oxytocin in 8 normal mares and 6 mares with delayed uterine clearance to determine if intrauterine fluid stimulated prostaglandin release. Mares received 2 treatments in a cross-over design. Treatment 1 consisted of 20 IU, i.v., oxytocin during estrus. Treatment 2 consisted of an infusion of 10 mL, i.u., saline 15 min prior to oxytocin administration. Treatments were performed 4 to 6 h apart. Blood was collected and PGFM was measured as in experiment 1. Data were analyzed by least squares analysis of variance. In Experiment 1, regression analysis of scintigraphy and PGFM profiles indicated that time response curves differed between groups (P < 0.01). At 120 min, normal mares retained 40.4 +/- 4.9% (mean +/- SEM) of the radiocolloid while mares with delayed clearance retained 88 +/- 5%. Fifteen minutes after oxytocin administration (135 min), all normal mares and 4 of 5 mares with delayed clearance retained only < 6% of the colloid. During the first 120 min, plasma PGFM concentrations did not differ between the 2 groups. After oxytocin was given, plasma PGFM concentrations increased in 4 of 5 mares with delayed uterine clearance (80 to 3,096 pg/mL) but not in normal mares (13 to 46 pg/mL). In Experiment 2, plasma PGFM concentrations did not rise in normal mares but rose in 3 of 6 mares with delayed clearance (135 to 483 pg/mL) independent of treatment or period. The results suggest that intrauterine clearance of radiocolloid after oxytocin administration appears to be independent of PGF2 alpha release in normal mares during estrus. The difference in prostaglandin release response after oxytocin administration between the 2 groups was unrelated to the presence of intrauterine fluid.

Human chorionic gonadotropin-induced alterations in ovarian follicular dynamics during the estrous cycle of heifers.

This experiment was designed to characterize hCG (3,000 IU)-induced alterations in ovarian follicular and corpus luteum (CL) dynamics during the estrous cycle (EC) in heifers. Following synchronization of estrus (norgestomet implant for 7 d with injection of PGF2alpha 1 d before implant removal), 13 heifers were treated with either hCG (n = 6) or saline (control group; n = 7) on d 5 of the EC (d 0 = day of estrus). Blood sampling from the jugular vein and ultrasonography of both ovaries were conducted daily until confirmation of ovulation following a detected estrus. Treatment with hCG at d 5 induced formation of an accessory CL in all hCG-treated heifers. Subsequent plasma progesterone (P4) concentrations were higher (P < .01) between d 9 and 17 for the hCG group than for the control group. More (P < .05) hCG-treated heifers had EC with three waves of follicles than control heifers. The second-wave dominant follicle (DF) emerged earlier in hCG than in control heifers (7.3 vs 10.4 d; P < .01). A group x wave interaction (P < .01) was detected for duration of the second and third wave follicles; hCG treatment decreased duration of the second wave (6.3 vs 9.3 d) and increased duration of the third wave (9.2 vs 5.3 d). The hCG-treated heifers had preovulatory follicles that lasted longer (P < .01) on the ovary than those in control heifers. However, plasma estradiol concentrations did not differ (P > .10) between hCG-treated and control heifers during the preovulatory period. A wave x duration interaction (P < .01) was detected for P4 concentrations, between d 13 and 22, for heifers with two vs three spontaneous-wave EC. Higher P4 concentrations during the luteal phase may contribute to a greater rate of follicular turnover or frequency of three-wave cycles, and lower P4 concentrations were associated with two-wave cycles.

Induction of abortion in the bitch with a synthetic prostaglandin analog.

Eight mixed-breed bitches in the middle (30 to 35 days) and late 3rd (53 to 56 days) of gestation were given 20 microg of synthetic prostaglandin analog/kg of body weight, IM (4 bitches) or the polyethylene glycol vehicle, IM (4 bitches). Bitches treated with prostaglandin aborted 2 days after treatment, whereas bitches given the vehicle delivered live pups at term. Luteolysis occurred within 24 hours of treatment, as shown by rapidly declining plasma progesterone concentrations. Microscopic signs of luteal degeneration were seen in ovaries removed 5 days after treatment. Three bitches erroneously designated as pregnant were treated during anestrus and had no marked changes in plasma progesterone or in gross and microscopic appearance of the reproductive tract. Serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased slightly in 4 bitches on the day after treatment, but values declined to base-line values 24 hours later. Treatment side effects included panting, salivation, vomiting, repeated defecation, and transient pain at the injection site.

Efforts to induce estrus in the bitch, using pituitary gonadotropins.

Induction of fertile estrus in 14 anestrous bitches was attempted, using pituitary gonadotropins given by intramuscular injection in 3 treatment regimens: as a single dose of 10-mg follicle stimulating hormone (FSH; 5 bitches), as multiple doses of FSH (1, 2, 4, 8, 16 mg) given to 4 bitches on days 1 and 2, 3 and 4, 5 and 6, 7 and 8, and 9 and 10, and as multiple doses of combined FSH and luteinizing hormone (LH; 1:1, 1:1, 2:1, 4:1, 8:2.5, 16:5 mg) given to 5 bitches on days 1, 3, 5, 7, 9, and 11, respectively. Four bitches were injected with equivalent volume of isotonic NaCl solution (saline). Treatments were evaluated by observation of sexual behavior in presence of a male, microscopic changes in exfoliated vaginal epithelium, plasma concentrations of estradiol-17 beta and progesterone, and qualitative and quantitative, gross and microscopic changes in the ovaries and uterus. Four of the 14 treated bitches came into estrus and mated (one became pregnant), two after single, and two after multiple doses of FSH. In each of the four, vaginal cornification, plasma estradiol and progesterone patterns, and ovarian and uterine changes were comparable with those seen in the one saline-treated bitch with a spontaneous estrous cycle and pregnancy. Three treated bitches manifested proestrous behavior (2 after a single injection and 1 after multiple injections of FSH) but had no measurable response in the vaginal epithelium, endocrine patterns, or appearance of ovaries and uterus. Seven gonadotropin-treated bitches and 3 saline-treated bitches did not respond in any way.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of a five-day progesterone-based timed AI protocol to determine if flunixin meglumine improves pregnancy per timed AI in dairy heifers.

Two experiments were conducted to test the hypothesis that the 5 d Co-Synch + CIDR (Controlled Internal Drug Release insert containing progesterone) protocol could be applied as an efficient timed AI (TAI) protocol in dairy heifers, and that treatment with flunixin meglumine (FM) during the period of CL maintenance would increase pregnancy per TAI (P/TAI) and late survival of embryos. Objectives were: 1) in Experiment 1, to compare P/TAI with the 5 d Co-Synch+CIDR protocol to a PGF(2alpha)/GnRH protocol; and 2) in Experiment 2, to determine if FM administered 15.5 and 16 d after first TAI would increase P/TAI, using the 5 d Co-Synch+CIDR protocol with a new or previously used (5 d) CIDR insert. In Experiment 1, 248 heifers were assigned randomly to either the PGF(2alpha)/GnRH protocol (n=120) or the 5 d Co-Synch+CIDR protocol (n=128). Pregnancy per TAI did not differ between the 5 d Co-Synch+CIDR protocol (53.1%) and the PGF(2alpha)/GnRH protocol (45.8%; P=0.22). In Experiment 2, 325 heifers synchronized with the 5 d Co-Synch+CIDR protocol were assigned randomly to receive two injections of FM (FM group; n=158) at 15.5 and 16 d after TAI, or to remain as untreated controls (n=165). Pregnancy per TAI in Experiment 2 was 59.4 and 59.5% at 45 d for control and FM groups, respectively, with no differences between groups (P=0.83). The 5 d Co-Synch+CIDR protocol resulted in an acceptable P/TAI in dairy heifers. However, FM did not improve P/TAI in dairy heifers.

Relationship between endometrial oxytocin receptors and oxytocin-induced prostaglandin F2 alpha release during the oestrous cycle and early pregnancy in pony mares.

The effect of transcervical endometrial biopsy on the concentrations of plasma immunoreactive oxytocin and 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM) was studied in 18 pony mares on days 8, 12 and 14 after ovulation, days 12 and 14 of early pregnancy and at oestrus. Five biopsy specimens were taken within 15 min and consecutive specimens from each mare were pooled two (A) and three (B) together for measurement of the number of oxytocin receptors. Blood samples were collected at intervals of 5 min for 15 min beginning just before the initial biopsy. Biopsy procedure elicited prompt oxytocin release in all mares. Pregnancy did not affect the response but day after ovulation had a significant influence on oxytocin release. The greatest increase in plasma oxytocin was observed on day 12 in both nonpregnant and pregnant mares and the lowest on day 8. The concentration of plasma PGFM rose linearly over the 15 min period in nonpregnant mares. This response increased progressively with time after ovulation and was greatest on day 14. There was no increase in circulating PGFM in pregnant mares. Endometrial oxytocin receptor concentration was lowest in mares at oestrus and highest in nonpregnant mares on day 14. Oxytocin receptor density in pregnant mares was similar to that in nonpregnant mares on day 12 but was significantly attenuated on day 14. The affinity of oxytocin receptors was lower in pregnant than in nonpregnant mares. Because of the positive correlation between PGF2 alpha release, endometrial oxytocin receptor density, and plasma oxytocin concentrations in nonpregnant mares, it is assumed that the release of PGF2 alpha was induced by oxytocin and was mediated by oxytocin receptors. Pregnancy-induced inhibition of PGF2 alpha release was not associated with suppression of oxytocin release or oxytocin receptor density. An embryo-derived factor is therefore the most likely cause for the suppression of PGF2 alpha release and interruption of the oxytocin-PGF2 alpha interaction in mares during early pregnancy.

Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and immunolocalization of proteins synthesized by dog endometrium and membranes.

Previous studies on cyclic and pregnant bitches on dioestrus days 3-10 indicated synthesis de novo of at least ten protein complexes, two of which are major protein groups (cP5, M(r) 54,600; cP6, M(r) 23,000). Protein expression differed by day but not by status, suggesting that the embryo does not affect uterine protein synthesis. Ovariectomized bitches treated with various steroid regimens showed induction of cP5 and cP6 only after oestrogen priming followed by progesterone. Recently, N-terminal amino acid microsequencing has identified cP6 as a member of the retinol-binding protein (RBP) family. The present study was designed (a) to identify and characterize proteins synthesized de novo in explant culture from cyclic (dioestrus days 10-16) and early pregnant (dioestrus days 10-26) dogs, (b) to examine distribution of proteins by endometrium preimplantation (PI), and between (BI) and at (I) implantation sites; (c) to characterize proteins synthesized by embryonal membranes and (d) to localize RBP and CUPED (a cat oestrogen-dependent uterine glycoprotein) immunocytochemically in the uterus and embryonal membranes. Proteins synthesized by endometrium were examined by incorporation of [3H]leucine or [35S]methionine on two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by fluorography or autoradiography. Nine proteins, characterized by electrophoresis and previously found between dioestrus days 3-10, appeared to be expressed through day 16 of cyclic and day 26 of pregnant dioestrus, although many decreased and cP5 was lost after day 13. In BI and I sites, RBP decreased in number of isoelectric variants as gestation progressed, while the major M(r) form (23,000) was reduced to 21,500. There was no change in M(r) and isoelectric variants of RBP from cyclic dogs, suggesting differences in RBP gene regulation. Proteins synthesized by embryonal membranes appeared to be serum proteins. Immunolocalization of RBP confirmed that oestrogen priming followed by progesterone was required for induction. In pregnant bitches, staining was present in all luminal epithelia on dioestrus day 10, but by day 17 only specific epithelium stained. As pregnancy progressed to day 24 of dioestrus, staining was localized to specific epithelial cells of the deep spongy zone and yolk sac. In ovariectomized bitches, CUPED was confirmed to be oestrogen dependent with staining in both luminal and glandular epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)