Corpus luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG and progesterone

The effect of treatment with a GnRH agonist, hCG or progesterone (P(4)) on corpus luteum function and embryonic mortality was investigated in buffaloes inseminated during mid-winter. Italian Mediterranean buffaloes (n=309) were synchronized using the Ovsynch with timed-AI program and mated by AI at 16 h (Day 0) and 40 h after the second injection of GnRH. On Day 5, buffaloes were randomly assigned to four groups: Control (no treatment, n=69), GnRH agonist (buserelin acetate, 12.6 microg, n=73), hCG (1500 IU, n=75) and P(4) (PRID without E(2) for 10 days, n=77). Progesterone (pg/ml) was determined in milk whey on Days 5, 10, 15 and 20 and pregnancy diagnosis was undertaken on Day 26 by ultrasound and Day 40 by rectal palpation. Treatment with buserelin and hCG increased (p<0.05) P(4) on Day 15 compared with controls (456+/-27, 451+/-24 and 346+/-28 pg/ml, respectively). Buffaloes treated with a PRID had intermediate P(4) concentrations (380+/-23 pg/ml). Embryonic mortality between Days 26 and 40 (22.9%) and pregnancies at Day 40 (48.9%) did not differ between treatments. A higher (p<0.01) P(4) concentration was found on Day 20 in pregnant animals compared with non-pregnant and embryonic mortality buffaloes, which did not differ. In summary, buserelin and hCG increased P(4) concentrations on Day 15 but this was not associated with a reduced incidence of embryonic mortality in buffaloes during mid-winter.     

Influence of season on corpus luteum structure and function and AI outcome in the Italian Mediterranean buffalo (Bubalus bubalis)

The aim was to ascertain whether relationships between corpus luteum (CL) vascularization, CL function, and pregnancy outcome in AI in buffaloes were consistent across the breeding season and transition period to the nonbreeding season in a Mediterranean environment. Stage of the estrous cycle in Italian Mediterranean buffaloes was synchronized using the Ovsynch with timed AI program and buffaloes were mated by AI in both the breeding season (N = 131) and transition period (N = 125). Detailed investigation of CL structure and function was undertaken in 39 buffaloes at each of the respective times using realtime B-mode/color-Doppler ultrasonography on Days 10 and 20 after AI. Progesterone (P4) concentrations were determined by RIA in all buffaloes. Pregnancy rate on Day 45 after AI was greater (P < 0.05) during the breeding season (58.0%) than the transitional period (45.6%) and this was primarily the result of a lower (P < 0.05) late embryonic mortality during the breeding season (7.3%) compared with the transition period (23%). Circulating concentrations of P4 on Days 10 and 20 after AI were greater (P < 0.01) during the breeding season (4.6 ± 0.3 and 3.4 ± 0.2, respectively) than during the transition period (1.6 ± 0.12 and 1.8 ± 0.2, respectively), and this was independent of reproductive status as there was no interaction between pregnancy and season. Corpus luteum time average medium velocity at Day 10 after AI was greater (P < 0.01) during the breeding season (19.3 ± 1.5) than in the transitional period (8.3 ± 0.7). There were positive correlations in pregnant buffaloes between CL time average medium velocity and P4 concentrations on Day 10 (r = 0.722; P < 0.01) and Day 20 (r = 0.446; P < 0.01) after AI. The findings were interpreted to indicate that relationships between CL vascularization, CL function, and pregnancy outcome in AI in buffaloes are consistent across the breeding season and transition period to the nonbreeding season. The distinction between the breeding season and the transition period is the relatively low proportion of buffaloes that have CL function and P4 concentrations required to establish a pregnancy during the transition period, which is manifested in a greater incidence of embryonic mortality.     

Corpus luteum development and function and relationship to pregnancy during the breeding season in the Mediterranean buffalo

The aim of this study was to ascertain corpus luteum (CL) development and function in buffaloes synchronized and mated by artificial insemination (AI) during the breeding season. Italian Mediterranean buffalo cows (n = 43) at 86.5 ± 2.7 days postpartum were synchronized by the Ovsynch-TAI Program and inseminated using frozen thawed semen at 20 and 44 h after the second injection of GnRH. The CL dimensions (diameter and area) and blood flow were examined on Days 5, 10, 15, 20, and 25 after AI by realtime B-mode/colour-Doppler ultrasonography. The resistive index (RI), pulsatility index (PI) and time average medium velocity (TAMV) were recorded at each time, together with CL dimensions. Blood samples were taken on the days of ultrasonography for progesterone (P4) assay by RIA. Data were grouped into pregnant or non-pregnant and retrospectively analyzed by repeated measure ANOVA and correlation analyses. Dimensions of the CL on Days 10, 20, and 25 after AI were greater (P < 0.01) in buffaloes pregnant on Day 45 (n = 18) compared with non-pregnant buffaloes (n = 25). The former buffaloes also showed a greater (P < 0.01) rate of CL growth between Days 5 and 10 after AI. Blood flow to the CL on Day 10 after AI showed a higher TAMV (P < 0.01) and lower RI (P < 0.05) in pregnant buffaloes compared with non-pregnant buffaloes. Negative correlations were observed on Day 10 after AI between CL diameter and RI (r = −0.61; P < 0.01) and PI (r = −0.60; P < 0.01); P4 concentrations and RI (r = −0.46; P < 0.02); and RI and pregnancy (r = 0.45; P < 0.02). Positive correlations were observed between pregnancy and CL size (r = 0.54; P < 0.01), ΔCL diameter between Days 5 and 10 (r = 0.52; P < 0.01), ΔCL area between Days 5 and 10 (r = 0.48; P < 0.015), and ΔP4 between Days 5 and 10 (r = 0.50; P < 0.01). Based on these findings it is concluded that the period between Day 5 and 10 is very important for CL growth and crucial in evaluating pregnancy. Accordingly, the assessment of CL parameters during the period from Day 5 to Day 10 after AI might be used to predict the likelihood of an ongoing pregnancy.     

Evaluation of GnRH treatment 12 days after AI in the reproductive performance of dairy cows

The objective of this study was to evaluate the effects of GnRH administered at Day 12 post-AI on the reproductive performance of dairy cows. Holstein-Friesian dairy cows (n=103) on a large Hungarian dairy farm were allocated randomly to treated (n=54) or control (n=49) groups. Twelve days after AI, treated cows received a GnRH agonist i.m., while the control group received a placebo (physiological saline). Progesterone radioimmunoassay was used to determine the correct timing of artificial insemination (Day 0) and the incidence of luteal insufficiency on Day 12. Ultrasonography and radioimmunoassay for pregnancy-associated glycoprotein were used to detect pregnancy and late embryonic/fetal mortality between Days 32 and 55 after AI. Three cows from each group were inseminated when progesterone concentrations were >1.0 ng/mL, and six cows (four from the treated and two from the control group) had luteal insufficiency (progesterone<1.0 ng/mL) on Day 12. Late embryonic/fetal mortality occurred in three treated cows and in two control cows. When these cows were removed from the model, calving rates after first service were 59.6% (28/47) and 59.1% (26/44) for treated and control cows, respectively (P>0.05). There was no significant difference between treated and control cows when they were inseminated before or after Day 100 from calving. In summary, administration of a GnRH agonist on Day 12 after AI did not improve reproductive performance in dairy cows. However, our approach may be used for the field evaluation of different treatment protocols.     

Delayed treatment with GnRH agonist, hCG and progesterone and reduced embryonic mortality in buffaloes

The present study examined the effect of delayed treatment with tropic hormones and progesterone (P₄) on embryonic mortality in buffaloes. Buffaloes with a conceptus on Day 25 after AI were assigned to the following treatments: Control (n = 41), i.m. physiological saline; GnRH agonist (n = 36), i.m. 12 μg buserelin acetate; hCG (n = 33), i.m. 1500 IU hCG; P₄ (n = 38), i.m. 341 mg P₄ every 4 days on three occasions. Control buffaloes had an embryonic mortality of 41.4% (17/41) between Days 25 and 45, and this was reduced (P < 0.01) by treatment with GnRH agonist (11.1%, 4/36), hCG (9.0%, 3/33) and P₄ (13.1%, 5/38). On Day 45, buffaloes treated with hCG and which ovulated had greater (P < 0.05) concentrations of P₄ in whey (453 ± 41 pg/ml) than buffaloes in the same treatment that did not ovulate (297 ± 32 pg/ml). A similar but non-significant trend was observed for buffaloes treated with GnRH agonist. It was concluded from the findings that the treatment of buffaloes on Day 25 after AI with tropic hormones or P₄ is beneficial to processes associated with embryonic implantation.     

Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a qualitative and quantitative milk progesterone test

We compared three methods for diagnosing early pregnancy in cattle: 1) a trans-rectal ultrasound scan of the uterus, 2) a cow-side enzymeimmunoassay (EIA) milk progesterone test 3) a radioimmunoassay (RIA) milk progesterone test. Scanning of the uterus was performed in 148 cows. These cows were not detected in estrus before scanning, which took place between Days 21 and 33 after insemination (AI). A considerable difference was noted between the reliability of the scannings performed at an early stage (Days 21 to 25) and those performed at a later stage (Days 26 to 33). The sensitivity and specificity of the ultrasound examination between Days 21 and 25 were only 44.8% and 82.3%, respectively, but were 97.7% and 87.8% between Days 26 and 33, respectively. Milk samples were collected on the day of AI. (Day 0) and 21 days later. Samples that were positive in the EIA test always contained more than 1 ng/ml progesterone (P4); however, 20% of the negative EIA samples contained also more than 1 ng/ml P4. Only 59% of the animals showing a negative EIA test on Day 0 and a positive test on Day 21, indicating pregnancy, calved, while 16% of the cows with a negative test on Day 0 and Day 21, indicating nonpregnancy, turned out to be pregnant. Of the 82 animals with P4 levels lower than 1 ng/ml on Day 0 and higher than 1 ng/ml on Day 21, only 61.0% calved. All 14 cows with low levels both on Day 0 and Day 21, indicating nonpregnancy, were found to be not pregnant. The influence of both early embryonic death and the accumulation of intrauterine fluids on the accuracy of these tests are discussed.     

Seasonal fluctuations in the response of Italian Mediterranean buffaloes to synchronization of ovulation and timed artificial insemination

A comprehensive study of the efficiency of synchronization of ovulation and timed artificial insemination (TAI) was undertaken in a large group of Italian Mediterranean buffaloes at a commercial dairy. A total of 2791 synchronization protocols were carried out on 857 animals over 3 years. Of these protocols, 823 (29.5%) did not proceed beyond Day 7 (due to the absence of a vascularized CL) and 620 (22.2%) were discontinued on Day 10 (due to the absence of follicles >1.0 cm and tonic uteri); hence, 1443 (51.7%) protocols did not progress to TAI. Data were analyzed for four periods: P1, transition to spring (from breeding season to low breeding season); P2, low breeding season; P3, transition to fall (low breeding season to breeding season); and P4, breeding season. No differences were found among the four periods in terms of the proportion of protocols that did not result in TAI. Of the 857 buffaloes, 660 (77%) conceived and delivered a calf. The average number of TAI per pregnancy was 2.1 and ranged from 1.9 to 2.3 across years. Logistic regression analysis showed that buffaloes that calved during P3 had a higher odds ratio for pregnancy (1.380; P < 0.05) than buffaloes that calved in other periods. Pregnancy was also influenced by the calving to service period (odds ratio = 0.977; P < 0.01) and the pregnancy per AI (P/AI) at successive TAI (odds ratio = 1.480; P < 0.01). The pregnancy per AI at the first TAI (424/857, 49.5%) was greater (P < 0.01) than in subsequent TAI. The occurrence of late embryonic mortality (between Days 27 and 45 after TAI) was similar among the four periods. These findings indicated that there are distinct seasonal differences in the response of Italian Mediterranean buffaloes to synchronization and TAI.     

Use of bovine pregnancy-associated glycoproteins to predict late embryonic mortality in postpartum Nelore beef cows

The primary objective was to determine if circulating concentration of bovine pregnancy-associated glycoproteins (bPAGs) on Day 30 after artificial insemination (AI) may serve as a marker of late embryonic mortality in Bos indicus (Nelore) beef cows. In experiment 1, postpartum Nelore beef cows (n = 56) were artificially inseminated at a fixed time (Day 0) after synchronization of ovulation. Serum samples were collected on Days 0, 21, 24, 27, and 30 after AI. The first significant increase (P < 0.0001) in serum bPAGs after insemination occurred on Day 24 of gestation. In experiment 2, ovulation was synchronized in postpartum Nelore beef cows (n = 1460) and AI was received at a fixed time. Pregnancy diagnosis and blood sample collection were carried out on Days 28 to 30 after insemination. Cows that maintained a pregnancy from Days 28 to 100 of gestation (n = 714) had significantly (P < 0.0001) higher circulating concentrations of bPAGs on Day 28 compared with cows that did not maintain a pregnancy (embryonic mortality [EM]) until Day 100 (n = 89). When Day 28 bPAG concentration was included in a logistic regression model to predict pregnancy maintenance until Day 100 of gestation, there was an increase (P < 0.0001) in the probability of maintaining pregnancy as maternal concentrations of bPAGs increased. A receiver operating characteristic curve was generated to determine bPAG concentrations on Day 28 that should predict embryonic survival or mortality with an accuracy of 95% or more. On the basis of the positive and negative predicative value analysis, at Day 28 of gestation a circulating concentration of bPAGs greater than 7.9 ng/mL was 95% accurate in predicting embryonic maintenance (to Day 100); a concentration of bPAGs less than 0.72 ng/mL was 95% accurate in predicting EM by Day 100. In experiment 3, the preceding model was tested in a separate set of Nelore beef cows to validate whether bPAGs would serve as an accurate measure of late embryonic mortality. Ovulation was synchronized in 650 Nelore cows and received AI at a fixed time. Pregnancy diagnosis and bPAG sampling were performed at Day 28 of gestation. Only pregnant cows were included in the analysis. On the basis of the previously reported bPAG cutoff values, the test was 95% accurate in predicting late embryonic mortality at Day 28 of gestation. In summary, bPAGs seem to be a good marker for predicting EM between Days 28 and 100 of gestation and suggest that this model could help dissect the molecular mechanisms leading to late EM.     

Reproductive performance of dairy cows following treatment with cloprostenol 26 and/or 40 days postpartum: a field trial

One hundred and seventy Holstein Friesian cows were randomly assigned to receive either 500 ug cloprostenol or saline placebo on Day 26 postpartum followed by 500 ug cloprostenol or saline on Day 40 postpartum. Four treatment groups were formed: Group 1 - saline (Day 26)/saline (Day 40); Group 2 - cloprostenol/(Day 26) saline (Day 40); Group 3 - saline (Day 26)/cloprostenol (Day 40); Group 4 - cloprostenol (Day 26)/cloprostenol (Day 40). Double blind techniques were used in administering treatments and in assessing the response to treatment. Palpation of the reproductive tract per tectum and uterine biopsies were performed on 92 cows prior to each treatment at Day 26 and Day 40 postpartum. Progesterone concentrations were determined on milk samples collected prior to treatment. There were no significant differences among treatment groups with respect to services per conception, number of heats detected before first service and culling for infertility. Cloprostenol treatment at Day 26 appeared to delay the first estrus, but it reduced the number of days to conception after the first service. Cows receiving cloprostenol at Days 26 and/or 40 had a decreased calving-to-conception interval compared to controls (P = 0.01). Sequential therapy with two doses of cloprostenol resulted in slightly better reproductive performance than either treatment on Day 26 or 40 alone. Treatment with cloprostenol resulted in a decrease in the subsequent incidence of pyometra (P < 0.05). It is concluded that in the herd studied, cloprostenol therapy at Day 26 and/or 40 postpartum was beneficial to reproductive performance. Although it was anticipated that cloprostenol would be more effective in cows with elevated progesterone levels, the opposite was observed at the Day 26 cloprostenol treatment. Uterine biopsy at Days 26 and/or 40 had a detrimental effect on subsequent reproductive performance.     

Comparison of two Ovsynch protocols (GnRH versus LH) for fixed timed insemination in buffalo (Bubalus bubalis)

We evaluated the efficiency of replacing GnRH with LH in the ovulation synchronization protocol in buffaloes. Buffaloes received GnRH on Day 0, (Buserelin; Conceptal, 20 microg), PGF2alpha (Luprostiol; Prosolvin, 15 mg) on Day 7 and GnRH (Buserelin; Conceptal, 10 microg; Group 1) or porcine LH (LH; Lutropin-V, 12.5 mg; Group 2) on Day 9. In Experiment 1, we studied the follicular dynamics of 30 buffaloes (Group 1, n = 15 and Group 2, n = 15). We performed ultrasonography every 12 h from Days 0 to 2, then on Day 7 and then every 6 h from the time of GnRH or LH treatment (Day 9) until the time of ovulation. All females not ovulating by 48 h after the second GnRH or LH injection were considered as nonresponders. In Experiment 2, we evaluated 305 buffaloes (Group 1, n = 154; Group 2, n = 151), using the same two treatments studied in Experiment 1. We also recorded and evaluated aspects like parity, lactational status, the presence of mucus, and uterine tone at the time of artificial insemination (Al). In Experiment 1, ovulation rate after the first GnRH was 86.6% (26/30). Ovulation rates were 93.3% (14/15; Group 1) after the second dose of GnRH and 93.3% (14/15) after LH (Group 2). Ovulation occurred 36.4+/-10.4 h after the first GnRH. The interval for treatment to ovulation was 26.5+/-9.6 h for buffaloes treated with GnRH (Group 1) and 24.4+/-7.9 h for buffaloes treated with LH (Group 2); the time of ovulation did not differ statistically between the two groups (GnRH versus LH; P > 0.05). In Experiment 2, conception rates of the animals AI in the field were 56.5% (Group 1) and 64.2% (Group 2), respectively (P = 0.08). The response to the treatment with LH was not different to the treatment with GnRH; however, multiparous buffaloes had higher conception rates than the primiparous buffaloes in both groups (P > 0.05). Buffaloes with mucus at the time of AI in Group 2 had higher conception rates than the buffaloes that had mucus in Group 1 (P < 0.05). Uterine tone and lactational status did not influence conception rates (P > 0.05). In summary, the results showed that both treatments resulted in synchronization of ovulation and acceptable conception rates. Therefore, the exogenous injection of LH can substitute the GnRH injections in the Ovsynch program in buffaloes.     

Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 microg GnRH on Day 0, 375 microg PGF(2alpha) on Day 7 and 100 microg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID) containing 1.55 g progesterone and a capsule with 10mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF(2alpha) and 1000 IU PMSG at the time of PRID withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF(2alpha) and PMSG injections (Group B). Progesterone (P(4)) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P(4) concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P(4) (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P(4) 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P(4) on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID, particularly in cyclic buffalo.     

Luteotrophic effect, growth and survival of whole versus half embryos and, their relationship with plasma progesterone concentrations of recipient dairy heifers

This prospective and randomised experiment was designed to compare the luteotrophic effect of whole versus half embryos and, to evaluate the relationship between the plasma progesterone (P4) profiles and the rates of early embryonic (from Days 7 to 25), late embryonic (Days 25-42) and foetal (Days 42-63) mortalities of whole and half embryo recipients. Within a single herd, 188 virgin, healthy, cyclic, reproductively sound, with adequate body condition score, Holstein dairy heifers were randomly allocated to receive one whole or one half embryo on Day 7 of the oestrous cycle (Day 0=estrus). In each embryo-transfer (ET) group, half of the recipients were treated with a CIDR (controlled internal drug releasing device) between Days 7 and 19. Pregnancy was evaluated by ultrasound on Days 25, 42 and 63 and plasma P4 profiles were obtained until Day 63 of pregnancy. CIDR-treated and untreated heifers had similar pregnancy rates on Days 25, 42 and 63 and, embryo size on Day 42 was also similar in treated and untreated recipients. Therefore, CIDR treatment failed to promote growth and survival of half and whole embryos. Half embryos presented a significantly higher rate of early and late embryonic mortality than whole embryos. In contrast, foetal mortality was similar in whole and half embryos and, this was coincidental to a similar embryo size on Day 42. Therefore, half embryos exhibited a compensatory growth until Day 42, irrespective of CIDR treatment, after which they presented a similar survival rate to that of whole embryos. Half embryo-derived pregnancies presented significantly lower plasma P4 concentrations on Day 25 than whole embryo-derived pregnancies, suggesting that this lower luteotrophic effect of half embryos could be related to their higher rate of late embryonic mortality. No significant relationship between the early luteal P4 concentrations and embryo survival was observed in whole and half embryo recipients. The first detectable luteotrophic effect of embryonic origin was observed on Day 14 and no detectable second luteotrophic effect was observed until Day 63 of pregnancy. Treatment with CIDR significantly increased plasma P4 concentrations during treatment but induced a significant decrease after removal of the device, suggesting that secretion of luteotropins was downregulated in the course of treatment.     

Delayed treatment with GnRH agonist,hCG and progesterone and reduced embryonic mortality in buffaloes

The present study examined the effect of delayed treatment with tropic hormones and progesterone (P₄) on embryonic mortality in buffaloes. Buffaloes with a conceptus on Day 25 after AI were assigned to the following treatments: Control (n = 41), i.m. physiological saline; GnRH agonist (n = 36), i.m. 12 μg buserelin acetate; hCG (n = 33), i.m. 1500 IU hCG; P₄ (n = 38), i.m. 341 mg P₄ every 4 days on three occasions. Control buffaloes had an embryonic mortality of 41.4% (17/41) between Days 25 and 45, and this was reduced (P < 0.01) by treatment with GnRH agonist (11.1%, 4/36), hCG (9.0%, 3/33) and P₄ (13.1%, 5/38). On Day 45, buffaloes treated with hCG and which ovulated had greater (P < 0.05) concentrations of P₄ in whey (453 ± 41 pg/ml) than buffaloes in the same treatment that did not ovulate (297 ± 32 pg/ml). A similar but non-significant trend was observed for buffaloes treated with GnRH agonist. It was concluded from the findings that the treatment of buffaloes on Day 25 after AI with tropic hormones or P₄ is beneficial to processes associated with embryonic implantation.     

Determination of early pregnancy and embryonic growth in goats by transrectal ultrasound scanning

Ultrasonography has been shown to be a useful tool for pregnancy diagnosis and the study of embryonic growth in mammals. The objectives of this study were 1) to evaluate the use of real-time B-mode ultrasonography for early pregnancy diagnosis in goats, 2) to define criteria for accurate diagnosis of pregnancy, and 3) to monitor the embryonic growth ultrasonically until Day 40 after mating. Estrus was synchronized in 16 cyclic Anglo-Nubian goats with a single injection of cloprostenol (125 micrograms, i.m.). Estrous females were randomly assigned into 2 groups: 1) goats mated by a vasectomized male (n = 5; MV group), and 2) goats mated by an intact male of proven fertility (n = 11; MF group). Transrectal ultrasonographic examinations with a 5 MHz linear array transducer were performed from Days 13 to 40 post mating. The evaluated parameters included the appearance of nonechogenic areas in the uterus, presence of embryo(s), crown-rump length of embryo and embryonic heart rate (beats/min). On Day 18, the mean (+/- SEM) diameter of nonechogenic areas was 1.5 +/- 0.3 mm in the MV group and 4.0 +/- 0.5 mm in the MF group (P < 0.01). In 36% of the pregnant does these areas were less than 3 mm. The mean (+/- SEM) day of the first detection by means of heartbeats of at least 1 embryo was 20.7 +/- 0.5 d (range, Days 19 to 23). From Days 19 to 38 of pregnancy, crown-rump length was best represented by a linear regression (Y = -2.23 + 0.13X; r2 = 0.94; P < 0.05). Crown-rump length on the day of the first detection of an embryo was 5.3 +/- 0.3 mm, reaching 34.2 +/- 0.6 mm on Day 40. Mean (+/- SEM) heartbeat rate was 168.3 +/- 2.8 beats/min on Day 21, decreasing to 158.3 +/- 2.0 beats/min on Day 40. Detection of the caprine embryo by ultrasonography and confirmation of its viability by heartbeats was shown to be a reliable method for early pregnancy diagnosis in Anglo-Nubian goats. Ultrasonic measurement of crown-rump length was useful in predicting the age of the embryo.     

Evaluation of false ultrasonographic pregnancy diagnoses in sows by measuring the concentration of unconjugated estrogens in feces

On Days 26, 28, and 30 after AI, ultrasonographic pregnancy diagnoses were performed on 207 gilts and sows by using a 3.5 MHz linear-array transducer. Fecal samples were taken from the rectum after each ultrasonographic examination, and the concentrations of unconjugated estrogens in selected samples (n = 73) were measured by RIA. Fecal unconjugated estrogen concentration of 11.7 ng/g feces or higher was indicative of pregnancy. The overall sensitivity and specificity of the ultrasonographic test was 99% for farrowing sows and 73.1% for nonfarrowing sows. With one exception, sows with a false negative diagnosis by ultrasonography on Day 26 were correctly diagnosed pregnant by elevated fecal unconjugated estrogens or repeated ultrasonographic examinations on Days 28 or 30. Return to estrus around the sampling period may cause false positive results in the unconjugated estrogen assay, while early embryonic mortality can result in false positive diagnoses in both the ultrasonographic test and estrogen assay. Although there was a positive correlation between the concentrations of unconjugated estrogens in the feces and litter size at farrowing in the selected sows, it seems very unlikely that fecal estrogens can provide an accurate tool for predicting litter size.     

Diagnosis of pregnancy by radioimmunoassay of a pregnancy-specific protein in the plasma of dairy cows

The accuracy and efficiency of progesterone (P4) and bovine pregnancy-specific protein B (bPSPB) radioimmunoassays (RIA) in detecting pregnant and nonpregnant dairy cows were compared at different stages of pregnancy. The study included 145 French Friesian heifers and cows from a single herd. A total of 175 artificial insemination (A.I.) and blood sampling procedures were performed. Animals were bled 24 d post AI for P4 RIA. They were bled at 24, 26, 30 to 35, and 70 +/- 9 after AI for bPSPB RIA. Females were declared nonpregnant when plasma P4 concentrations were lower than 1.5 ng/ml. With the bPSPB RIA, cows were nonpregnant when at least one of the B Bo x 100 replicates was higher than 95% in the RIA. When compared with palpations per rectum at 70 d, the accuracy of positive diagnoses (no. positive and pregnant/no. positive diagnoses) by P4 RIA at Day 24 was 67.2% (82 122 ). The accuracy of negative diagnoses was 98% (52 53 ). Accuracy of positive diagnoses by bPSPB RIA increased with gestation age (P<0.05) from 86.2% (50 58 ) on Day 24 to 98.8% (83 84 ) at time of palpation per rectum. Accuracy of negative diagnoses increased (P< 0.001) from Day 24 (71.8%; 84 117 ) to Days 30 to 35 (100%, 83 83 ). Efficiency in detecting nonpregnant females was much higher (P < 0.001) with the bPSPB RIA on Days 30 to 35 (90.2%; 83 92 ) than with the P4 RIA on Day 24 (56.5%, 52 92 ). It is concluded that 30 days after AI, the bPSPB RIA is an efficient test both for pregnancy prediction and detection of nonpregnant dairy cows.     

Fertilization and early embryonic development in androstenedione-immunized Merino ewes

Ewes were immunized against androstenedione (Fecundin) and assigned to be mated 14 days (179 ewes Group C) or 25 days (174 ewes Group B) after a booster immunization with Fecundin. The anti-androstenedione titres at these times were 6790 and 3240 respectively (P less than 0.01). The remaining 169 ewes were untreated controls (Group A). Ewes were mated to entire rams (12 rams to 180 ewes) at their second oestrus after synchronization of oestrus. Immunization against androstenedione caused a shortening of the time from sponge removal to mating (Day 0) and a decrease in the percentage of ewes mated by the rams. Also, ovulation rate was increased after immunization (P less than 0.01), being 1.42, 2.16 and 1.93 for Groups A, C and B respectively. Egg recovery rates on Day 2 were lower in immunized ewes and there was some indication that fertilization rates were lowered. On Day 13 after mating a higher proportion of blastocysts was recovered from ewes in Group A than from those in Groups B and C. Immunization resulted in lower fertilization rates and smaller blastocysts with lower mitotic indexes (P less than 0.01). At Days 24-32 of pregnancy fetal weight was lower in the immunized ewes. At all sampling stages, the proportion of ewes pregnant (fertility) was lowered in immunized ewes. The results of the present study show that significant reproductive wastage occurs in androstenedione-immunized Merino ewes, with lower rates of embryo recovery and delayed embryonic development being found in comparison to controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcervical artificial insemination in sheep: effects of a new transcervical artificial insemination instrument and traversing the cervix on pregnancy and lambing rates

Cervical anatomy limits the use of transcervical intrauterine artificial insemination (TC AI) in sheep. We have developed an instrument to cope atraumatically with the cervix; although this instrument has not affected fertilization rate or pregnancy rate through Day 3, the effects on sperm transport and pregnancy after Day 3 are not known. The objective of the present study was to determine whether our TC AI instrument affected sperm transport, pregnancy rates, or lambing rate. In Experiment 1, ewes were assigned to two treatments: TC AI using the new TC AI instrument (n=10) or AI via laparotomy using a laparoscopic AI instrument (n=10). Twenty hours after artificial insemination, the uterine horns and oviducts were recovered and flushed to collect spermatozoa. Sperm transport did not differ (P>0.05) between the two treatments. In Experiment 2, ewes were assigned to three treatments: TC AI using the new TC AI instrument+sham intrauterine AI via laparotomy (n=29); sham TC AI+intrauterine AI via laparotomy using a laparoscopic AI instrument (n=29); and sham TC AI+intrauterine AI via laparotomy using the new TC AI instrument (n=30). On Day 14 after AI, uteri were collected and flushed to recover blastocysts. Transcervical deposition of semen reduced (P<0.05) Day 14 pregnancy rate (17.2% versus 61%), but intrauterine deposition of semen using the TC AI instrument via midventral laparotomy increased (P<0.05) Day 14 pregnancy rate (76.6% versus 44.8%). In Experiment 3, ewes were assigned to two treatments: sham cervical manipulation (n=40) or cervical manipulation to mimic TC AI (n=40). Immediately after treatment, each ewe was mated with a ram and watched until the ram mounted and ejaculated into the ewe. Treatment did not affect Day 30 or 50 pregnancy rate (67.5 and 66.2%, respectively), determined ultrasonically, or lambing rate (62.5%). The differences between Days 30 and 50 pregnancy rates and lambing rate were not significant. In Experiment 4, ewes were assigned to two treatments: TC AI (n=99) or laparoscopic AI (n=99). Transcervical AI reduced (P<0.01) Day 30 (TC AI versus laparoscopic AI; 5.0% versus 46.0%) and Day 50 pregnancy rates (4.0% versus 41.0%), determined ultrasonically, and lambing rate (4.0% versus 41.0%). Although the TC AI procedure significantly reduced pregnancy and lambing rates, large numbers of spermatozoa deposited at natural insemination seemed to compensate. Because our TC AI procedure has all but eliminated any visual evidence of trauma, and because the procedure does not seem to affect sperm transport or embryonal survival until Day 3, we speculate that cervical manipulation associated with TC AI may activate pathways that interrupt pregnancy between Days 3 and 14.     

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography.In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of Day 21 samples, 41% of Day 25, 95% of Day 35, 96% of Day 42, 99% of Day 49 and in 100% of samples of Days 56 and 63. Concentrations of PSPB increased (P < 0.05) from Days 21 to 42 and from Days 56 to 63, with a plateau between Days 42 to 56. Demi embryo pregnancies had higher (P < 0.05) plasma PSPB concentrations on Days 35 and 42 than whole embryo pregnancies. Progesterone supplementation had a positive effect (P < 0.01) on PSPB concentrations from Days 35 to 63. Concentrations of PSPB were similar in non-supplemented whole and demi embryo pregnancies from Days 7 to Day 63. In contrast, in supplemented recipients, demi embryo pregnancies had higher (P < 0.05) PSPB concentrations on Days 25 to 42 than whole embryo pregnancies. No significant correlation was found between P4 and PSPB concentrations or between the concentrations of these hormones and embryonic measurements on Day 42. In conclusion, demi embryos experienced a compensatory growth until Day 42 of pregnancy, attaining a similar size to that of whole embryos and originating conceptuses producing similar plasma PSPB concentrations to those of whole embryo derived conceptuses. Embryonic growth and conceptus secretion of PSPB were positively stimulated by early pregnancy exogenous P4 treatment.     

The extent and timing of prenatal loss in gilts

The potential litter size of gilts that is based on the ovulation rate is much higher than the actual litter size, which depends on the fertilization rate and subsequent prenatal mortality. Prenatal mortality is divided into embryonic mortality (before Day 30) and fetal mortality (after Day 30). Prenatal loss includes both fertilization failure and prenatal mortality. Crossbred gilts (n = 149) were bred at the first observed estrus after being exposed to the boar at 200 days of age. Time of the first insemination after estrus detection was determined by measurement of vaginal conductivity using a Walsmeta meter. A second insemination was administered either 8 or 16 hours later. Artificial insemination with fresh semen (0 to 3 days old) was used throughout the experiment. Gilts were slaughtered on Day 3 (n = 26), Day 10 (n = 42), Day 30 of gestation (n = 45) or they were allowed to farrow (n = 36). Gilts slaughtered on Day 3 were used to estimate the fertilization rate. Gilts slaughtered on Day 10 and Day 30 were used to calculate embryonic mortality, while fetal mortality was calculated from the gilts that farrowed. The mean (+/-SEM) number of corpora lutea (CL) was 13.15+/-0.46, 13.36+/-0.37 and 12.97+/-0.39 for gilts slaughtered at Days 3, 10 and 30, respectively (P>0.05), and the mean (+/-SEM) number of normal embryos recovered was 11.12+/-0.69, 9.46+/-0.55 and 9.33+/-0.58, respectively. Litter size at parturition was 9.10+/-0.54. There was a significant difference between the number of normal embryos on Day 3 and Day 30 (P=0.05) and also between the number of normal embryos at Day 3 and the number of piglets at term. Ninety percent of the ova were recovered at Day 3. The fertilization rate was calculated either 1) assuming that unrecovered ova had a similar fertilization rate as the recovered ova (FRER=94.5+/-2.0%) or 2) assuming that unrecovered ova were unfertilized (FROR=84.5+/-2.5%). It was concluded that FRER was a more accurate estimation of the fertilization rate. Based on this fertilization rate, embryonic mortality between Day 3 and Day 10 was 20.8+/-8.3%, with an additional 12.5+/-7.1% loss between Day 10 and Day 30, when all gilts were included (P = 0.308). Thus the total prenatal loss, including fertilization failure, up to Day 10 was 26.3% and to Day 30 it was 38.8%. Fetal mortality was 2.2%, giving a total prenatal mortality (excluding fertilization failure) of 35.5% and a prenatal loss of 41%. Most of the prenatal loss was due to embryonic mortality. In those gilts that remained pregnant most of the embryonic loss occurred before Day 10 (19.0+/-6.3%; P=0.003). There was no further loss between Day 10 and 30 of pregnancy. There was a significant difference between the loss from Day 3 to Day 10 compared with the loss from Day 10 to Day 30 (P=0.05); therefore, most of the embryonic loss in pregnant gilts occurred before Day 10. Since fetal mortality was 3.2+/-6.3%, most of the prenatal loss was due to embryonic mortality.