| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,4
* Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-0201;
and
North Central Research and Outreach Center, University of Minnesota, Grand Rapids 55744-3396;
and
Northwest Research and Extension Center, Kansas State University, Colby 67701-0786; and
and
Agricultural Research Center-Hays, Kansas State University, Hays 67601-9228
2 Correspondence:
Dept. of Anim. Sci. and Ind., Manhattan 66506-0201 (phone: 785-532-1243; fax: 785-532-7059; E-mail:
jss{at}ksu.edu).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
(PGF2; Cosynch) or with Cosynch plus a 7-d treatment with an intravaginal progesterone (P4)-releasing insert (CIDR-B; Cosynch + CIDR). In Exp. 2, 183 lactating beef cows were treated with the Cosynch protocol or with Cosynch plus a 7-d treatment with norgestomet (Cosynch + NORG). In Exp. 1 and 2, blood samples for later P4 analyses were collected on d -17, -7 (first GnRH injection), 0 (PGF2 injection), and at timed artificial insemination (TAI; 48 h after PGF2). In Exp. 3, 609 lactating beef cows were treated with the Cosynch + CIDR protocol or were fed 0.5 mg of melengestrol acetate (MGA) per day for 14 d before initiating the Cosynch protocol 12 d after the 14th d of MGA feeding (MGA + Cosynch). Blood samples were collected as in Exp. 1 and 2, plus additional samples on d -33 and -19 before PGF2. In Exp. 4, 360 lactating beef cows were treated with a Cosynch + CIDR protocol, with TAI occurring at either 48 or 60 h after PGF2, while receiving either GnRH or saline to form four treatments. Blood samples were collected as in Exp. 1 and 2. In Exp. 1, addition of P4 reduced the ability of the first GnRH injection to induce ovulation in anestrous cows with low P4 before PGF2 but improved (P = 0.06) pregnancy rates (61 vs 66%). In Exp. 2, the addition of NORG mimicked P4 by likewise increasing (P < 0.01) pregnancy rates (31 vs 51%) beyond those after Cosynch. In Exp. 3, the Cosynch + CIDR protocol increased (P < 0.001) pregnancy rates from 46 to 55% compared to the MGA + Cosynch protocol. In Exp. 4, administration of GnRH at TAI improved (P < 0.05) pregnancy outcomes (50 vs 42%), whereas timing of TAI had limited effects. We conclude that a progestin treatment concurrent with the Cosynch protocol improved pregnancy outcomes in all experiments, but pretreatment of cows with MGA was not as effective as the CIDR insert or NORG implants in this Cosynch-TAI model. Most of the improvement in pregnancy rates was associated with the increase in pregnancy rates of anestrous cows, regardless of whether ovulation was successfully induced in response to GnRH 7 d before PGF2. Injection of GnRH at TAI following the Cosynch + CIDR protocol increased pregnancy rates in cycling cows with high P4 before the PGF2 injection and in anestrous cows with low P4 before PGF2 injection.
Key Words: Artificial Insemination • GnRH • Progestogen • Prostaglandins • Synchronization
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
(PGF2) injection followed by detected estrus and AI (Thompson et al., 1999; Stevenson et al., 2000). The Cosynch protocol is similar with the addition of a second GnRH injection given at 48 h after PGF2, at the time of one fixed timed AI (TAI; Stevenson et al., 2000; Geary et al., 2001a). These protocols increase pregnancy rates in noncycling suckled cows (Thompson et al., 1999; Stevenson et al., 2000) and couple induced ovulation and follicular maturation with luteal regression in anestrous and cycling cows (Twagiramunga et al., 1995; Thompson et al., 1999). Feeding melengestrol acetate (MGA) before administering two injections of PGF2 11 d apart (Patterson et al., 1995) or before Select Synch (MGA Select; Patterson et al., 2000) also has improved pregnancy rates.
Combining GnRH and a progestin results in fewer cows in estrus just before or early after the injection of PGF2 and fewer cows induced to ovulate have short estrous cycles subsequent to treatment with GnRH plus a progestin (Thompson et al., 1999). Addition of a progestin may be essential to maximize pregnancy rates in cows at risk to not conceive during the breeding season (e.g., those that calve late, that are young, and/or in poor body condition; Stevenson et al., 2000; Lamb et al., 2001).
The objectives of the first three experiments were to determine 1) the ability of GnRH to induce cycling activity in anestrous cows with or without a concurrent progestin treatment, and 2) whether pregnancy rates are greater after providing a source of progestin either before (via feeding of MGA) or concurrent (via norgestomet (NORG) or progesterone [P4]) with the Cosynch protocol. The objectives of the fourth experiment were to optimize timing of AI after the Cosynch protocol in which P4 was utilized and to determine the necessity of the second injection of GnRH administered after TAI to initiate an LH surge for maximal pregnancy rates.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Following spring calving in 1998, purebred lactating beef cows located at the Kansas State University Purebred Beef Unit in Manhattan were maintained on a mixture of dormant and vegetative native pastures. Until pastures developed, cattle were supplemented with protein and either prairie or alfalfa hay to meet or exceed NRC (1996) recommendations for cows nursing calves. Numbers of cows, breed, parity, body condition, and days postpartum at the onset of the breeding season are summarized in Table 1
. Cows were paired according to breed, parity (primiparous vs multiparous), and days postpartum, and pairs were assigned randomly to each of two treatments (Figure 1). Cows were administered (i.m.) one 100--g injection of GnRH (Fertagyl; Intervet Inc., Millsboro, DE) 7 d before (d -7) and another 48 h after a 25-mg injection (i.m.) of PGF2 (d 0; Lutalyse; Pharmacia Animal Health, Kalamazoo, MI). One TAI was administered 48 h after PGF2 just prior to the second GnRH injection (Cosynch; n = 92). A second treatment was similar, but cows also received one intravaginal P4 insert (controlled internal drug release; CIDR-B, InterAg, Hamilton, NZ; 1.9 g of P4) during the 7 d between the first injection of GnRH and PGF2 (Cosynch + CIDR; n = 95).
|
|
injection), and at 48 h. The d 0 blood sample was collected between 1 and 2 h after the CIDR insert removal. Samples were placed on ice, held overnight at 5°C, and allowed to clot. Blood samples were centrifuged (2000 x g) for 15 min, sera were harvested, and then frozen until concentrations of P4 were quantified. Multiple sires and technicians were balanced across treatments. Pregnancy was diagnosed by transrectal ultrasonography 34 to 35 d after the TAI, in which fluid in uterine horns, presence of embryo, or both were detected. Cows were not exposed to clean-up bulls but were inseminated after detected estrus during a 45- to 60-d breeding season. Experiment 2
Following spring calving in 1999, purebred lactating beef cows were located at the Kansas State University Purebred Beef Unit and maintained on a mixture of dormant and vegetative native pastures. Feeding of cows was as described for Exp. 1. Numbers of cows, breed, parity, body condition, and days postpartum at the onset of the breeding season are summarized in Table 1
. Cows were paired according to breed, parity (primiparous vs multiparous), and days postpartum, and pairs were assigned randomly to each of two treatments (Figure 1). Cows in one group received the Cosynch protocol described above for Exp. 1 (Cosynch; n = 91). A second treatment was similar, but cows also received one hydroin ear implant containing 6 mg of NORG (Syncro-Mate-B, Merial Ltd., Iselin, NJ) during the 7 d between the first injection of GnRH and PGF2 (Cosynch + NORG; n = 92).
Blood samples were collected on d -17, -7, 0 and at 48 h, as described in Exp. 1. The d 0 blood sample was collected at the same time as the NORG implant was removed from the ear. Multiple sires and technicians were used. Pregnancy was diagnosed and breeding season duration were as described for Exp. 1. Body condition scores (BCS; Whitman, 1975; 1 = thin and 9 = fat) were assessed on d 0 of the experiment.
Experiment 3
This study was conducted at four locations: University of Minnesota North Central Research and Outreach Center, Grand Rapids; DarLynn Ranch, Pierz, MN; Kansas State University Purebred Beef Unit; and Thielen Ranch, Dorrance, KS. Numbers of cows, breed, parity, body condition, and days postpartum at the onset of the breeding season are summarized in Table 1.
Cows were blocked by parity (primiparous vs multiparous), paired by days postpartum at the onset of the breeding season within breed (where applicable), and pairs were assigned randomly to each of two treatments (Figure 1). In the first treatment, cows were fed with 0.5 mg of MGA for 14 d starting 33 d before the beginning of the breeding season (d 0). The feeding period was followed by the Cosynch protocol described above, beginning 12 d after withdrawal of MGA from the feed (MGA + Cosynch; n = 315). The GnRH product used was Cystorelin at a 100--g dose (Merial, Iselin, NJ). In a second treatment, cows were treated with the Cosynch protocol plus a CIDR (CIDR-B; InterAg, Hamilton, NZ) containing 1.38 g of P4 for 7 d (d -7 to 0; Cosynch + CIDR; n = 294).
Cows were maintained on native pastures and supplemented with either sorghum silage or prairie hay. The diets of cows at the Kansas locations were supplemented with 1.8 kg of a ground or pelleted grain-base formulation containing either 0 or 0.5 mg MGA·cow-1·d-1 during a 14-d feeding period. Cows were accustomed to being fed in pasture bunks before MGA was introduced into the diet. At one of the locations where a pelleted mix was fed, all cows received the control pellets (Cattle Charge BT, MFA Agri Service, St. Joseph, MO) for 4 d before MGA in pellets (Cattle Charge ES, MFA Agri Service, St. Joseph, MO) was introduced for cows assigned to be fed MGA. At one Minnesota location (DarLynn Ranch), cows in dry lots were fed a total mixed diet that contained grass hay, corn, and soybean meal. During the 14-d feeding period cows received either 0 or 0.5 mg MGA·cow-1·d-1 mixed into 2.7 kg of grain and 0.4 kg soybean meal. At the University of Minnesota location, cows were fed in feed bunks located in pastures containing half legume and half native grasses. During the feeding period 0.9 kg of corn containing either 0 or 0.5 mg MGA·cow-1·d-1 was provided. Cows were managed as single groups in all locations except during the 14-d feeding period. In all locations, bunk space was not limiting.
Blood samples were collected on d -33, -19, -7, 0 (immediately upon removal of CIDR insert; Figure 1), and 48 h as described for Exp. 1 and 2. In addition, blood was collected on d 13 and 20 after the TAI. Blood samples on d -33 and -19 were not collected at one location due to distance and logistical limitations before application of treatments. Body condition scores were assessed on d 0 as in Exp. 2. Multiple sires and technicians were balanced across treatments. Pregnancy was diagnosed in all treated cows once between d 29 and 33 and again between d 54 and 61 after TAI. Diagnosis was by transrectal ultrasonography to visualize fluid, embryo, or both. At one location, the second pregnancy diagnosis was not performed because the cows were not available on the day of diagnosis. Clean-up bulls were withheld from cows for 10 d after TAI at the two private ranches, and the AI period continued for an additional 45 to 65 d at the two university locations.
Experiment 4
In Spring 2001, lactating beef cows were studied at two Kansas locations (Kansas State University Purebred Beef Unit and Agricultural Research Center-Hays; Table 1). At one location, cows were fed as described for Exp. 1 and 2. At the second location, cows were fed a base diet consisting of sorghum-sudan grass hay supplemented with oat hay and protein (26% CP) cubes to meet or exceed NRC (1996) requirements. A Cosynch + CIDR protocol was applied to all cows as described for Exp. 1 and 3. A previously used intravaginal P4 insert (CIDR-B) was inserted on d -7 and removed at the time of PGF2 administration (Figure 1). Cows were blocked by breed, calving date, parity, and assigned randomly to a 2 x 2 factorial arrangement of four treatments: 1) insemination beginning at 48 vs 60 h after PGF2 (d 0) and 2) administration of a second, 100--g injection of GnRH (Factrel, Fort Dodge Animal Health, Fort Dodge, IA) or an equivalent volume of saline immediately after TAI. Therefore, the four treatments were 48 h + GnRH, 48 h + saline, 60 h + GnRH, and 60 h + saline.
Blood samples were collected on d -17 (or -14), -7, 0 (immediately upon removal of the CIDR insert), and before timed insemination (48 or 60 h) for later analysis of P4 as described earlier. Body condition scores were assessed on d 0. Multiple sires were used and technicians performed all TAI using frozen-thawed semen equally applied to cows in both treatments. Pregnancy was diagnosed at d 35 to 36 after TAI as described previously. Clean-up bulls were withheld from cows for 10 d after TAI at one location, and the AI period continued for an additional 45 to 60 d at the second location.
This experiment was conducted during a time when availability of new CIDR inserts was limited so used CIDR inserts were employed in Exp. 4. The used CIDR contained either 1.38 or 1.9 g of P4 when new and had been used once or twice previously in lactating cows. Used inserts in Exp. 4 were applied randomly to cows. We (Richardson et al., 2002) have demonstrated in replacement beef and dairy heifers fitted used CIDR inserts in situ for 7 d and PGF2 given on d 6 that serum concentrations of P4 on d 8 averaged 1.5 ng/mL compared to 0.4 ng/mL in controls. These results indicate that adequate concentrations of P4 were produced by the used CIDR to meet the objective of Exp. 4 (provided during Cosynch protocol). We conducted Exp. 1 and 3 using new CIDR inserts under the authorization of the U.S. Food and Drug Administration Investigational New Animal Drug 6450. The intent of the current treatments in Exp. 4 was to test the efficacy of supplying P4 to prevent premature occurrence of estrus during the treatment period rather than testing the used CIDR insert itself, which could not be done without the concurrent administration of new CIDR inserts as controls. Application of the previously used CIDR inserts in no way implies that we endorse their reuse. Although precautions were taken to clean and sanitize the used CIDR inserts prior to their reuse, no guarantee of their purity, potency, or sterility can be made.
Radioimmunoassay of Progesterone
Progesterone was quantified in blood sera by RIA (Skaggs et al., 1986). Intra- and interassay CV were: 7.4% and 7%, respectively, for six assays (Exp. 1); 3.9% and 3.3%, respectively, for four assays (Exp. 2); 6.4 and 7.8%, respectively, for 24 assays (Exp. 3); and 6.8% and 7.5%, respectively, for 10 assays (Exp. 4).
Definitions
Induction of Estrous Cycles After MGA.
Concentrations of P4 on d -33, -19, and -7 (at three locations of Exp. 3 only) were used to evaluate whether ovulation was induced in noncycling cows in response to MGA treatment. When the first two samples (d -33 and -19) were low (noncycling) and the third was high (
1 ng/mL), then we concluded that MGA induced ovulation. In contrast, when concentrations of P4 were low (<1 ng/mL) in all three samples, then it was interpreted that MGA failed to induce ovulation.
Cycling Status Before First GnRH. Concentrations of P4 in blood sera collected on d -17 (or -14), and -7 (d -33, -19, and -7 in Exp. 3) were used to determine initial cycling status at the onset of the Cosynch protocol. Cows were classified as noncycling (anestrus) if all samples were low or as cycling if any of the samples were high.
Induced Corpus Luteum (CL) After First GnRH. Induction of a CL in anestrous cows after the first injection of GnRH was determined by concentrations of P4 on d -17 (or -14), -10, and 0 (d -33, -19, -7, and 0 for Exp. 3). If concentrations were low in all samples, then GnRH failed to induce ovulation. If the concentrations were low in all samples, but high at d 0, then GnRH induced ovulation and subsequent CL formation after its administration on d -7.
Cycling Status Before PGF2.
Cows included were those that had resumed estrous cycles before GnRH as well as those induced to cycle after GnRH on d -7 as described above.
Regression of GnRH-induced CL by PGF2.
Successful regression of the CL induced by GnRH was determined by samples collected on d -17 (or -19 for Exp. 3), -7, and 0. If P4 was low on d -17 (or -19) and d -7, but high on d 0 (indication of induced CL), and low at the TAI (48 or 60 h after d 0), then the induced CL was successfully regressed. If concentrations were low on d -17 (or -19) and d -7, high on d 0 (indication of induced CL), and high at TAI, then the induced CL did not regress after PGF2.
Presence of CL Before PGF2 Injection.
High concentrations of P4 on d 0 indicated the presence of a functional CL just before the PGF2 injection.
Regression of CL After PGF2.
The percentage of cows with a CL before PGF2 that subsequently regressed as a consequence of the PGF2 injection was determined by low concentrations of P4 at 48 h (or 60 h in Exp. 4). When concentrations were high on d 0 and low 48 to 60 h later, then the CL present on d 0 was regressed.
Synchronization Rate. Females with low or high concentrations of P4 on d 0 and low concentrations at TAI were defined as synchronized. This measure is nearly identical to the proportion of cows with low (<1 ng/mL) P4 at TAI. Based on this definition, anestrous cows with low P4 in all blood samples are classified as synchronized.
High P4 on d 13, 20, or Both, After TAI. These variables were defined as the proportions of cows with high P4 on d 13, 20, or both, after TAI (Exp. 3 and 4 only).
Pregnancy Rates. Pregnancy rates were defined as the total number pregnant after TAI divided by the total number of cows treated.
Embryo Survival. Embryo survival was defined as the proportion of cows that were first identified pregnant on d 29 to 33 that were still pregnant on d 54 to 61 after TAI (Exp. 3 only).
Miscellaneous.
Only in Exp. 1 was the CIDR insert removed between 1 and 2 h before blood was collected. This was not the case when the CIDR was removed in Exp. 3 and 4. As a result, high P4 at PGF2 could include cows with high P4 resulting from the CIDR alone and not a combination of CIDR and CL. Therefore, variables including induced CL after first GnRH, cycling status before PGF, regression of GnRH-induced CL by PGF, presence of CL before PGF, regression of CL after PGF, and synchronization rates in Exp. 3 and 4 may be overestimated because blood samples collected before PGF were in the presence of P4 released by the CIDR insert. A waiting period of 1 to 2 h was sufficient for concentrations of P4 in four ovariectomized cows after a 7-d insertion period to decrease from 2.8 ng/mL at the time of insert removal to 0.75 ng/mL by 1 h and 0.5 ng/mL by 2 h after insert removal from (Rathbone et al., 2002).
Statistical Analyses
Sires and AI technicians were applied randomly to cows within breed as much as possible in all experiments. Sires and technicians were different at each location and were therefore confounded with herd within experiment. When herd was included as a source of variation in any statistical model, the effects of sire and technician were part of the overall variation associated with herd.
All variables were analyzed by the GLM (continuous variables) and CATMOD (categorical or proportional variables) procedures (SAS Inst. Inc., Cary, NC) within experiment with one exception noted below. The basic model used to analyze all categorical variables defined above included treatment, herd or breed (where applicable), parity (primiparous vs multiparous), and two-way interactions with treatment plus days postpartum on d 0 and BCS as regression variables.
Pregnancy rates were analyzed with a model containing treatment, herd or breed (where applicable), parity, cyclicity on d 0 of the breeding season plus days postpartum as either a regression variable or as a fixed effect (<60, 60 to 80, or >80 d), and BCS as a regression variable or as a fixed effect (
4.5, 5.0, or 5.5). All two-way interactions with treatment were included except for the interaction of treatment x parity in the models for Exp. 3 and 4 because one location in each experiment had no primiparous cows.
Treatment or interaction percentages were separated using LSD tests when associated with a protected F-test (P < 0.05). Percentage means reported are the unadjusted (raw) percentages.
Further summary of pregnancy rates were made for all experiments to illustrate the effect of treatments on cows within various cycling and P4 patterns. Four categories of P4 patterns were created to determine their relationship with pregnancy rates. Cycling activity as defined above prior to GnRH administration on d -7 was further divided into those that had high (CL likely present) or low (no CL present) P4 on d 0 when PGF2 was administered to form four categories: cycling-high, cycling-low, anestrus-high, and anestrus-low. Within category, treatment differences were tested conservatively by the variance associated with herd nested within year (herd-year) using the mixed models procedure in SAS. Herd-year was declared to be random in the model.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Percentage of cows that had resumed estrous cycles before applying any treatment is shown in Table 1 for each parity group. A summary of results for Exp. 1 is in Table 2. The percentage of cows that were cycling exceeded 77% when assignments to each treatment occurred. Of those cows that were still anestrus on d -7, 2.6 times more (P < 0.01) Cosynch than Cosynch + CIDR cows had high P4 (induced CL) on d 0. Regression of this induced CL was equally effective (>92%) in both treatments. A tendency (P = 0.08) was detected for more Cosynch than Cosynch + CIDR cows to have high P4 on d 0 prior to injection of PGF2. The proportion of cows with high P4 (CL) on d 0 that had luteolysis after PGF2 (>57%) and synchronization rates (>94%) of all cows were not different between treatments. Concentrations of P4 in blood serum (Figure 2) did not differ between treatments on either d 0 or 2 (48 h when TAI occurred). Four of 119 (3.4%) cows (one Cosynch + CIDR cow) that became pregnant had concentrations of P4 1 ng/mL at TAI.
|
|
Experiment 2
Percentage of cows that were cycling before applying any treatment is shown in Table 1 for each parity group. Despite having calved on average more than 85 d before the onset of the breeding season (d 0), only 23% of the primiparous cows had resumed estrous cycles by d -7. Further results of this experiment are in Table 3. The percentage of cycling cows assigned to treatments approached 60%. Based on changes in blood P4 from d -7 to d 0, six times more (P < 0.01) anestrous cows in the Cosynch treatment responded to GnRH than responded to GnRH in the Cosynch + NORG treatment by having high P4 (induced CL) on d 0. Regression of this induced CL was equally effective (>95%) in both treatments. Considerably more (P < 0.01) Cosynch than Cosynch + NORG cows had high P4 on d 0 and more (P < 0.01) cows in the former than the latter treatment had luteolysis after PGF2. Synchronization rates exceeded 94% and were not different between treatments. Concentrations of P4 in blood serum were less (P < 0.05) in NORG-treated cows on d 0 but not on d 2 at TAI (Figure 2). Four of 119 (5.3%) cows (four Cosynch + NORG cows) that became pregnant had concentrations of P4 1 ng/mL at TAI.
|
Experiment 3
The proportion of primiparous and multiparous cows that had resumed estrous cycles before hormonal injections were administered on d -7 in this experiment are illustrated by herd in Table 1. In only one herd studied was the percentage of cows cycling <50%.
Comparisons of various reproductive characteristics for this experiment are summarized in Table 4. In three herds in which blood samples were collected on d -33 and -19, MGA did not increase the proportion of cows cycling before d -7. Blood samples were not collected on d -33 and -19 for the herd with the lowest percentage of cows cycling (42%; Table 1) and probably the greatest potential for MGA induction of estrous cycles. The percentage of cycling cows exceeded 89% on d -7. Addition of the CIDR insert tended (P = 0.07) to increase the proportion of anestrous cows with high P4 on d 0 compared to the MGA + Cosynch protocol. The proportion of cows with induced CL that regressed after PGF2 on d 0 was not different between treatments, but more (P < 0.001) CIDR-treated cows had high P4 on d 0. Although the proportion of cows in which luteolysis occurred was greater (P < 0.001) in the CIDR treatment, synchronization rates were similar.
|
). In contrast to the previous two experiments, 53 of 298 (17.8%) cows (24 MGA + Cosynch and 29 Cosynch + CIDR cows) that became pregnant had concentrations of P4 1 ng/mL at TAI.
Despite the greater proportion of cows with elevated P4 at TAI, pregnancy rates for Exp. 3 were acceptable (Table 5). Pregnancy rates for the Cosynch + CIDR treatment were superior (P < 0.001) to those of MGA + Cosynch treatment regardless of how pregnancy rates were assessed (based on high concentrations of P4 on d 13 and 20, on d 20, or by transrectal ultrasonography on d 29 to 33). Although herd was a significant (P 0.05) source of variation in pregnancy rates, the CIDR treatment produced greater pregnancy rates in three of four herds. Neither days postpartum, parity, nor BCS affected pregnancy rates. Overall, fewer anestrous cows tended (P = 0.11) to conceive after the TAI than those cows that were cycling before d -7. Although pregnancy rates were less in anestrous than cycling cows, a significant proportion of anestrous cows conceived nonetheless. Of cows in each treatment in which ovulation was likely induced after GnRH, 11 of 19 (58%) anestrous cows conceived after Cosynch + CIDR compared to seven of 19 (37%) cows after MGA + Cosynch. Further, of those anestrous cows in which P4 was never elevated before TAI, one of one cow conceived in the Cosynch + CIDR treatment compared with zero of five cows after Cosynch + CIDR.
|
Experiment 4
In this experiment, the same ovulation synchronization protocol was applied to all cows in two herds, Cosynch + CIDR. The proportion of cows that had resumed estrous cycles before the modified Cosynch + CIDR protocol was applied is summarized in Table 1. Cyclicity of one herd was about 77% of the other. Comparisons of various reproductive traits in primiparous and multiparous cows are summarized in Table 6. The proportion of cows cycling before d -7 was less (P < 0.01) in multiparous cows, which calved 24 to 33 d later than the primiparous cows. Percentage of anestrous cows with high P4 (induced CL) on d 0 also was less (P < 0.01) in mature cows. The percentage of induced CL that regressed was not different between parity groups. Fewer (P < 0.05) multiparous than primiparous cows had high P4 on d 0 and luteolysis by 48 to 60 h after PGF2. Despite those differences, the synchronization rates (>97%) were not different between parity groups.
|
. Three of 167 (1.8%) cows (all Cosynch + CIDR cows) that became pregnant had concentrations of P4 1 ng/mL at TAI. Although the proportion of cows with high P4 at 13 and 20 d after TAI was not different between parity groups, concentrations of P4 in pregnant cows were greater (P < 0.05) than those of nonpregnant cows on d 13 and 20 (Figure 2).
Pregnancy outcomes are summarized in Table 7. Timing (48 vs 60 h) of TAI did not affect pregnancy rates, but the injection of GnRH improved (P < 0.05) pregnancy rates compared to saline (no interaction of main effects was detected). Cycling but not anestrous cows benefitted from the GnRH injection at TAI. Anestrous cows inseminated at 48 h had similar pregnancy outcomes to anestrous cows inseminated at 60 h, whereas cycling cows numerically, but not significantly, fared better when TAI occurred at 60 than 48 h. Neither herd, days postpartum, cycling status, nor parity influenced pregnancy rates. In contrast, interactions of hormone and time with BCS tended (P < 0.10) to occur. Cows with BCS of 5 or more tended (P = 0.07) to benefit from the GnRH injection at TAI compared to those with BCS 4.5. Cows with BCS of 5 or less tended (P < 0.10) to have greater pregnancy rates when TAI occurred at 60 than 48 h.
|
. Cycling status before the onset of the Cosynch + CIDR protocol was paired with whether the cow had high P4 (presence of a CL) or low P4 on d 0 when PGF2 was administered. An interaction (P = 0.06) tended to occur between P4 pattern and treatment. When AI occurred at 48 or 60 h, cycling cows with high P4 on d 0 and anestrous cows with low P4 on d 0 had better pregnancy outcomes when the additional GnRH injection was administered after TAI. Too few cycling cows had low P4 on d 0 to determine the best treatment combination. However, anestrous cows with high P4 on d 0 (those with induced ovulation after the first GnRH injection on d -7) had better pregnancy outcomes when inseminated at 60 h without the additional GnRH injection.
|
Comparisons of pregnancy outcomes resulting from all experiments are illustrated in Figure 3. Among cows that had resumed estrous cycles (cycling) before the onset of hormone injections on d -7 and had high P4 on d 0 or among those in which concentrations of P4 were low on d 0, no treatment protocol was superior. Addition of the CIDR insert or the NORG implant produced the best pregnancy rates among anestrous cows, whether ovulation was induced successfully after GnRH on d -7. For anestrous cows with low P4 on d 0 that were treated with Cosynch + CIDR treatment, pregnancy rates tended (P = 0.06) to be greater than contemporary anestrous cows treated with the Cosynch protocol. In general, across all categories, the Cosynch + CIDR or Cosynch + NORG treatments consistently produced the best pregnancy outcomes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
One of the greatest limitations of such programs is that too few suckled cows have resumed normal estrous cycles before the onset of the breeding season. Protocols that include a GnRH injection given 6 or 7 d before PGF2 have partly overcome this limitation because GnRH induces ovulation (Twagiramungu et al., 1995; Thompson et al., 1999; Stevenson et al., 2000). Further success was achieved when a progestin is incorporated into the treatment protocol (Stevenson et al., 1997a; Thompson et al., 1999; Stevenson et al., 2000) because it normalizes uterine secretions and prevents a short luteal phase that often follows GnRH- or hCG-induced ovulations in postpartum cows (Inskeep, 1995; Thompson et al., 1999).
Cyclicity
The percentage of multiparous cows that had resumed estrous cycles generally was greater in all experiments despite the fact that primiparous cows calved on average up to 24 d before the cow herd. Ranges in the percentages of cows that were cycling varied from 23 to 98% among primiparous cows and 42 to 100% among mature cows across all five herds during 4 yr. Days postpartum at the onset of the breeding season, age at calving, body condition at calving or later, and nutritional conditions are factors known to influence intervals to first estrus and subsequent pregnancy rates (Short et al., 1990; Stevenson et al., 1997b). Specifically, reestablishment of postpartum estrous cycles in cows with BCS < 5 (Whitman, 1975) is delayed by postpartum dietary energy restriction.
Pregnancy outcomes after TAI in our studies were consistently greater in cycling than anestrous cows. Results from all experiments, regardless of treatment protocols, indicated that pregnancy rates were at or near 50% except in Exp. 2 where the percentages of cycling cows were significantly less in the primiparous than multiparous cows despite having calved 13 d earlier on average. When the proportion of cycling cows in a herd was 70%, the pregnancy rates after TAI were near 50% or better.
Induction of Ovulation and Subsequent Luteolysis
The response of anestrous cows to GnRH whether preceded by (e.g., MGA in Exp. 3) or concurrent with (P4 in Exp. 1 and 4 or NORG in Exp. 2) progestin treatment was investigated in these studies. In Exp. 1 and 2, only 13 to 25% of the anestrous cows were induced to ovulate after GnRH in the presence of either the CIDR insert or NORG implant compared to 67% of anestrous cows treated with only GnRH. These results are consistent with other research that showed that beginning the feeding of MGA on the same day of the GnRH injection reduced incidence of induced ovulations from that expected for cycling heifers treated on d 5 and 18 of the estrous cycle (Pancarci et al., 1999). In contrast to results in Exp. 1 and 2, in Exp. 3 (GnRH + new CIDR) and 4 (GnRH + used CIDR), 95 and 70% of anestrous cows were induced to ovulate, respectively. The high incidence of induced ovulation in Exp. 3 and 4 after the CIDR treatment must be interpreted with caution because of the possibility that P4 was merely elevated due to the CIDR insert and not as a result of more cows ovulating and forming a CL following GnRH administration. These observations are consistent with the fact that the CIDR was removed between 1 and 2 h before blood collection in Exp. 1, whereas CIDR inserts were removed at the time of blood collection in Exp. 3 and 4 and may have contributed to this greater proportion of cows with elevated P4 on d 0. The difference in the proportion of anestrous cows that ovulated after GnRH did not seem to be related to the amount of P4 released by a new or previously used CIDR insert. Further, concentrations of P4 on d 0 seemed to be greater in cows of Exp. 3 than those in Exp. 1 in which new CIDR inserts also were used and those in Exp. 4 where used CIDR inserts were employed. We (Richardson et al., 2002) have reported that intact heifers with a used CIDR insert in situ for 7 d and PGF2 on d 6 had concentrations of P4 in blood serum that averaged 1.5 ng/mL 24 h after PGF2, whereas those of intact heifers without a CIDR averaged 0.4 ng/mL. These results indicate that the used CIDR released sufficient P4 to account for blood concentrations of approximately 1 ng/mL.
Treatments involving single or multiple injections of GnRH have been used to induce ovulation in noncycling cows (Troxel and Kesler, 1984; Crowe et al., 1993; Troxel et al., 1993). More than 80% of anestrous cows treated with GnRH plus a progestin implant (NORG) ovulated and formed their first CL observed via ultrasonography and verified by elevated blood P4 (Thompson et al., 1999). The combination of GnRH and progestins increased follicular development and proportion of early first postpartum ovulations in suckled beef cows compared to treatments with GnRH alone (Thompson et al., 1999).
Induced cycling activity after MGA was not observed in Exp. 3 probably because in the three herds tested, cycling activity was quite high (Table 3). Studies designed to determine the efficacy of MGA to induce cyclicity in cows are limited. Progestin treatments were effective in inducing ovulation and behavioral estrus in prepubertal heifers (Jaeger et al., 1992) and anestrous cows (Patterson et al., 1995; Fike et al., 1997). Studies with P4 (CIDR) alone not only improved synchronization of estrus but also initiated estrus and ovulation in a percentage of prepubertal heifers (Lucy et al., 2001) and anestrous cows (Mcmillan and Macmillan, 1989; Fike et al., 1997; Lucy et al., 2001). The latter studies imply that secretion of LH increased during and after progestin treatment in postpartum anestrous cows and prepubertal heifers reflected an elevation in pituitary storage of LH. In prepubertal heifers, the increase of LH also may be the result of progestins accelerating the prepubertal decline in estradiol negative feedback on LH through site specific reductions in the number of neurons containing estradiol receptors in the hypothalamus. A similar mode of action for progestins also may occur in postpartum cows (Fike et al., 1997).
In all experiments, more than 89% of the CL induced by GnRH regressed when PGF2 was administered 7 d after GnRH. These observations confirm our earlier observations when NORG was used in conjunction with GnRH (Thompson et al., 1999) and with P4 (CIDR) plus GnRH (Lamb et al., 2001).
Luteolysis after PGF2
The percentages of cows with PGF2-induced CL regression varied among experiments. Percentages of luteolysis were similar among CIDR-treated cows in Exp. 3 (75%) and 4 (78%), both of which were greater than that observed in Exp. 1 (58%). These differences are partly explained by the fact that in Exp. 1 the CIDR inserts were removed between 1 and 2 h before blood sampling occurred to allow the CIDR contribution to concentrations of serum P4 to decrease. Further, in all experiments where P4 was administered via the CIDR insert, concentrations of P4 were not different between treatments 48 or 60 h after CIDR insert removal. Thus, the differences in experiments were likely related to the residual effects of P4 contributed by the CIDR inserts. However, in Exp. 2, when NORG was used, an equally poor (42%) rate of luteolysis occurred as in Exp. 1 with the CIDR (58%). Because pregnancy rates were greater after these treatments that employed progestin in the form the CIDR or NORG, these poorer rates of luteolysis seemed to be of little importance to pregnancy outcomes.
Synchronization Rates
Synchronization rates exceeded 90% in Exp. 1, 2, and 4, but were just over 80% in Exp. 3. These rates were nearly identical to the proportion of cows with P4 < 1 ng/mL at TAI. Unexplainably, synchronization rates in the Cosynch + CIDR treatment of Exp. 3 seemed to be less than those rates in Exp. 1, 2, and 4. Concentrations of P4 seemed to be greater in Exp. 3 on d 0 and at TAI (Figure 2). Although this was confirmed by a greater proportion of cows (17.8%) in Exp. 3 that had elevated P4 at TAI and yet became pregnant compared with the other experiments (<5.3%), pregnancy rates were not seemingly harmed.
High synchronization rates indicate that estrus and ovulation are likely closely linked to the timing of luteolysis. Combining GnRH and PGF2 is effective in synchronizing estrus and ovulation in beef cattle (Geary et al., 1998, 2001a,2001b; Stevenson et al., 2000). In those protocols, the first injection of GnRH induced ovulation or luteinization of the dominant follicle and subsequently initiated a new follicular wave (Twagiramungu et al., 1995; Thompson et al., 1999). The PGF2 injection ensured luteolysis before a second injection of GnRH induces ovulation approximately 24 to 34 h later (Pursley et al., 1995). After a second GnRH injection, 87 to 100% of treated cows ovulated within the next 32 h (Vasconcelos et al., 1999; Lamb et al., 2001). The ovulatory response is synchronized tightly and allows conception to occur when TAI is administered at the time of the second GnRH injection or 8 to 24 h later (Pursley et al., 1998; Geary et al., 2001b).
Luteal Function on d 13 and 20 After TAI
The proportions of cows with high P4 on d 13 after TAI in Exp. 3 and 4 consistently exceeded 93% in treatments with P4 via the CIDR insert, whereas those in MGA-treated cows were less. This difference was reflected by actual concentrations of P4 in Exp. 3 because concentrations of P4 were greater in pregnant cows previously treated with the CIDR insert compared to those fed MGA. In pregnant cows, the viable embryo enhances maternal P4 production as early as 6 to 10 d after estrus (Van Cleeff et al., 1991). Further, increased P4 in cows treated with a CIDR may be a carryover effect, whereby CL function was enhanced by prebreeding treatment of P4 plus GnRH.
Pregnancy Rates and Embryo Survival
Pregnancy rates were consistently improved by 5 to 20 percentage points in Exp. 1 and 2 when GnRH + either NORG or P4 was administered before injection of PGF2. In Exp. 3, P4 provided concurrently with the Cosynch protocol produced better pregnancy outcomes than pretreatment of cows with MGA before Cosynch in three of four herds. Those three herds had pregnancy rates >52% after the Cosynch + CIDR protocol. In Exp. 4, the Cosynch + CIDR protocol produced the best overall pregnancy outcomes when TAI occurred at either 48 or 60 h followed by GnRH injection. When considering cycling status and presence of a CL on d 0, in all cases except for anestrous cows with low P4 at d 0, pregnancy rates increased from 48 to 60 h when no GnRH was administered after TAI, indicating that optimal timing of AI after the Cosynch + CIDR protocol may well be between 48 and 60 h or later. For anestrous cows that failed to respond to the first GnRH injection and had no elevation in P4 before TAI, optimal timing may be closer to 48 than 60 h (Table 8).
Improved pregnancy rates are achieved when a progestin is incorporated into GnRH + PGF2 protocols between the first GnRH and the PGF2 injection in most (Stevenson et al., 1997a, 2000; Lamb et al., 2001) but not all studies (Johnson et al., 2000). Our experiments confirm the former reports for the benefit of the combination of GnRH with a progestin, particularly in anestrous cows. Pregnancy rates of suckled cows inseminated at detected estrus were greater after the Select Synch (GnRH injection 7 d before PGF2) plus NORG treatment protocol when compared to controls (two PGF2 injections 14 d apart) due to the greater pregnancy rates in noncycling cows in the former treatment (Thompson et al., 1999). Lactating beef cows pretreated with MGA before administering PGF2 also had greater pregnancy rates than controls treated only with PGF2 (Patterson et al., 1995).
In addition to the positive effect that P4 had for inducing ovulation (Mcmillan and Macmillan, 1989), greater pregnancy rate in the CIDR group can be attributed to other factors. Addition of progestins to an estrus synchronization protocol seems to be essential to maximize pregnancy rates among anestrous cows (Stevenson et al., 1997a, 2000; Lamb et al., 2001). Our results might indicate that the priming effect of P4 on the hypothalamic-pituitary axis to release endogenous GnRH, LH, and FSH and, consequently, induce cycling activity is more effective using the CIDR concurrently with ovulation synchronization than using MGA prior to the Cosynch protocol. In addition, as a result of increased concentrations of estradiol in cows previously treated with progestins, more LH was released after the GnRH injection (Thompson et al., 1999). Consistent improvements in pregnancy rates also may occur because fewer cows are in estrus just before or soon after the injection of PGF2. As a consequence, pregnancy rate is greater than in those without progestin because synchrony between AI and ovulation improves (Stevenson et al., 2000). After similar protocols, about 7% of dairy cows are observed in estrus before the PGF2 injection (Dejarnette et al., 2001).
Treatment with progestin could decrease the incidence of short luteal phases described in beef cows following first postpartum ovulation (Inskeep, 1995; Thompson et al., 1999). Pregnancy rates were consistently less in all anestrous cows treated with the Cosynch protocol (including those pretreated with MGA) when compared to those that were cycling. Perhaps with the later postpartum application of progestins (preCosynch treatment with MGA vs concurrent Cosynch treatment with the CIDR), cows are more likely to respond. It is likely that more of these anestrous cows had short estrous cycles after the Cosynch protocol. The incidence of short cycles was greater in cows treated with hCG in lieu of GnRH in conjunction with the latter protocol (Geary et al., 2001a). Short luteal phases after first postpartum estrus are a consequence of the premature release of uterine PGF2 that in turn causes premature CL regression and subsequent embryo death (Inskeep, 2000). Secretion of PGF2 increased during treatment of anestrous cows with progestins in the same manner as it did during the short luteal phase after gonadotropin injection in controls. Thus, without a previous exposure to P4, secretion of PGF2 increased prematurely when the first CL started to secrete P4, compromising fertility at that cycle (Inskeep, 1995).
Pretreatment of suckled cows with progestins resulted in normal fertilization and subsequent formation of a CL of normal life span in response to weaning (Inskeep, 1995) or GnRH injections (Thompson et al., 1999). Noncycling dairy cows induced to ovulate successfully and conceive after Select Synch and Ovsynch protocols (TAI 16 to 20 h after the second GnRH injection) had decreased ability to maintain those pregnancies after d 27 to 30 (Cartmill et al., 2001), but when similar treatments included P4 via the CIDR, embryo survival was increased relative to Ovsynch (El-Zarkouny et al., 2000; Pursley et al., 2001). Late embryonic death (between d 25 and 40 of gestation) has been estimated to be approximately 10% (Inskeep, 2000). Based on our reported embryo survival after d 29 to 33 until d 54 to 61, rate of embryo death (<10%) approximated those previous reports except for anestrous cows in the MGA + Cosynch protocol where losses were 23%.
Considering all four experiments, the best treatment to increase pregnancy rates in anestrous cows that had ovulated in response to GnRH on d -7 (high P4 on d 0) tended to be the concurrent treatment of a progestin with the Cosynch protocol. For those anestrous cows that did not respond to the first GnRH injection, administration of P4 via the CIDR produced the best pregnancy outcome. For cows that had resumed their estrous cycles before d -7 and had high P4 on d 0, no treatment was superior.
|
Implications |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
with timed artificial insemination at 48 h) increased pregnancy rates in three experiments. Pregnancy outcomes increased because more anestrous cows conceived, suggesting that the concurrent administration of progestin and GnRH are critical. A concurrent progestin treatment (CIDR) plus GnRH produced more pregnancies in three of four herds than feeding melengestrol acetate before the Cosynch protocol. However, the magnitude of this increase may not be sufficient to justify the cost of the CIDR compared to feeding melengestrol acetate. Insemination at either 48 or 60 h after the Cosynch + CIDR protocol produced similar pregnancy rates, indicating some flexibility in the insemination schedule. Because pregnancy rates and embryo survival were reflective of rates of cyclicity before the artificial insemination, managing body condition during late gestation and the calving season before the breeding season is critical.
|
Footnotes |
|---|
3 Dept. of Anim. Sci., Louisiana State University, Baton Rouge 70803-4210.
4 North West Research and Outreach Center, Crookston, MN 56716-5001.
Received for publication July 5, 2002. Accepted for publication October 7, 2002.
|
Literature Cited |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
estrus synchronization system. J. Anim. Sci. 70:2622–2627.[Abstract]
for ovulation control in postpartum suckled beef cows. J. Anim. Sci. 79:2253–2259. for synchronizing estrus and shortening the interval to pregnancy in postpartum beef cows, peripubertal beef heifers and dairy heifers. J. Anim. Sci. 79:982–995.. J. Anim. Sci. 73:954–959.[Abstract]
protocol. J. Anim. Sci. 78(Suppl. 1):218. (Abstr.)
, and GnRH. Theriogenology 44:915–923.
, and progesterone. J. Anim. Sci. 80:2792–2800. with or without timed insemination. J. Anim. Sci. 78:1747–1758.. J. Anim. Sci. 77:1823–1832.This article has been cited by other articles:
|
|
 |
|
  O. G. S. Filho, E. R. Vilela, T. W. Geary, and J. L. M. Vasconcelos Strategies to improve fertility in postpartum multiparous Bos indicus cows submitted to a fixed-time insemination protocol with gonadotropin-releasing hormone and prostaglandin F2{alpha} J Anim Sci, September 1, 2009; 87(9): 2806 - 2814. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Stevenson Progesterone, Follicular, and Estrual Responses to Progesterone-Based Estrus and Ovulation Synchronization Protocols at Five Stages of the Estrous Cycle J Dairy Sci, December 1, 2008; 91(12): 4640 - 4650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Schafer, J. F. Bader, J. P. Meyer, J. K. Haden, M. R. Ellersieck, M. C. Lucy, M. F. Smith, and D. J. Patterson Comparison of progestin-based protocols to synchronize estrus and ovulation before fixed-time artificial insemination in postpartum beef cows J Anim Sci, August 1, 2007; 85(8): 1940 - 1945. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. A. Perry, M. F. Smith, A. J. Roberts, M. D. MacNeil, and T. W. Geary Relationship between size of the ovulatory follicle and pregnancy success in beef heifers J Anim Sci, March 1, 2007; 85(3): 684 - 689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Stevenson, M. A. Portaluppi, D. E. Tenhouse, A. Lloyd, D. R. Eborn, S. Kacuba, and J. M. DeJarnette Interventions After Artificial Insemination: Conception Rates, Pregnancy Survival, and Ovarian Responses to Gonadotropin-Releasing Hormone, Human Chorionic Gonadotropin, and Progesterone J Dairy Sci, January 1, 2007; 90(1): 331 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Saldarriaga, D. A. Cooper, J. A. Cartmill, J. F. Zuluaga, R. L. Stanko, and G. L. Williams Ovarian, hormonal, and reproductive events associated with synchronization of ovulation and timed appointment breeding of Bos indicus-influenced cattle using intravaginal progesterone, gonadotropin-releasing hormone, and prostaglandin F2{alpha} J Anim Sci, January 1, 2007; 85(1): 151 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Stevenson, J. R. Pursley, H. A. Garverick, P. M. Fricke, D. J. Kesler, J. S. Ottobre, and M. C. Wiltbank Treatment of cycling and noncycling lactating dairy cows with progesterone during Ovsynch. J Dairy Sci, July 1, 2006; 89(7): 2567 - 2578. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Larson, G. C. Lamb, J. S. Stevenson, S. K. Johnson, M. L. Day, T. W. Geary, D. J. Kesler, J. M. DeJarnette, F. N. Schrick, A. DiCostanzo, et al. Synchronization of estrus in suckled beef cows for detected estrus and artificial insemination and timed artificial insemination using gonadotropin-releasing hormone, prostaglandin F2{alpha}, and progesterone J Anim Sci, February 1, 2006; 84(2): 332 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Bader, F. N. Kojima, D. J. Schafer, J. E. Stegner, M. R. Ellersieck, M. F. Smith, and D. J. Patterson A comparison of progestin-based protocols to synchronize ovulation and facilitate fixed-time artificial insemination in postpartum beef cows J Anim Sci, January 1, 2005; 83(1): 136 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Z. El-Zarkouny, J. A. Cartmill, B. A. Hensley, and J. S. Stevenson Pregnancy in Dairy Cows After Synchronized Ovulation Regimens With or Without Presynchronization and Progesterone J Dairy Sci, April 1, 2004; 87(4): 1024 - 1037. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Stevenson, S. K. Johnson, M. A. Medina-Britos, A. M. Richardson-Adams, and G. C. Lamb Resynchronization of estrus in cattle of unknown pregnancy status using estrogen, progesterone, or both J Anim Sci, July 1, 2003; 81(7): 1681 - 1692. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
