Pregnancy-associated glycoproteins (PAG) detection in milk samples for pregnancy diagnosis in dairy goats

In ruminants, glycoproteins synthesized in the outer layers of the trophoblast enter the maternal bloodstream. The assay of their concentration in plasma or in serum can be used for pregnancy diagnosis and to indicate feto-placental health status. In this study, concentrations of the pregnancy-associated glycoprotein (PAG) were investigated in the milk of dairy goats. The mean concentrations of PAG in milk were significantly higher in pregnant than in non-pregnant goats throughout the sampling period. Positive milk PAG levels were found in all pregnant animals from Day 32 after breeding, but remained at negative values in nonpregnant ones. This study reports for the first time a pregnancy diagnosis based on milk concentrations of PAG, that could be of great use on the farm.     

Risk factors for resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows

The objectives of this study were to evaluate factors associated with resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows. Holstein cows, 6396 from four dairy farms were evaluated to determine the relationships among parity, body condition score (BCS) at calving and at AI, season of year when cows calved, and milk yield on resumption of postpartum estrous cycles by 65 days postpartum, and all the previous variables, estrual or anestrus and AI protocol on conception rates and embryonic survival at the first postpartum insemination. Cows had their estrous cycle pre-synchronized with two PGF_2α injections given 14 days apart and were inseminated between 69 and 82 days postpartum following either an estrous or ovulation synchronization protocol initiated 12–14 days after the presynchronization. Blood was sampled and analyzed for progesterone twice, 12–14 days apart, to determine whether cows had initiated onset of estrous cycles after calving. Cows were scored for body condition in the week after calving, and again at AI, between 69 and 82 days postpartum. Pregnancy was diagnosed at 30 ± 3 and 58 ± 3 days after AI. Farm influenced all reproductive outcomes evaluated. More (P < 0.0001) multiparous than primiparous cows had initiated estrous cycles. Onset of estrous cycles was also influenced (P < 0.01) by BCS at calving and at AI, BCS change, season, and milk yield. More (P < 0.001) cows that had initiated estrous cycles than anestrous cows were pregnant at 30 and 58 days after AI, but anestrus did not affect pregnancy loss. Conception rates were also influenced (P < 0.01) by parity, BCS at calving and AI, BCS change, and season; however, milk yield and insemination protocol were not associated with conception rates at 30 and 58 days after AI. Factors that reduced conception rate on day 30 after AI also increased pregnancy loss between 30 and 58 days of gestation. Improving BCS at calving and AI, minimizing losses of BCS after calving, and hastening onset of estrous cycles early postpartum are all expected to increase conception because of enhanced embryonic survival.     

Early pregnancy diagnosis in sheep using near-infrared spectroscopy on blood plasma

The objective of this study was to evaluate the ability of near-infrared reflectance spectroscopy (NIRS) to discriminate between pregnant and nonpregnant ewes in early stages of pregnancy after artificial insemination (AI) from blood plasma. Samples were collected using jugular puncture at 18 and 25 days after AI from 188 Rasa Aragonesa and Ansotana ewes. Plasma samples were analyzed for pregnancy-associated glycoprotein (PAG) and progesterone (P4) using ELISA commercial kits. The spectra of plasma samples were recorded in the visible and near-infrared ranges. The performance of these tests were compared, using as criterion standard the pregnancy status determined using transabdominal ultrasonography at 45 days after AI. Pregnancy rate was 47.9% (90/188). At Day 18, sensitivity was similar in NIRS and P4 tests (98.9% vs. 100%; not significant) and greater than PAG (32.2%; both P < 0.001). Specificity was similar in NIRS and PAG tests (both 100%) and greater than that of P4 (84.7%; P < 0.001). At Day 25, sensitivity and specificity of NIRS and PAG were both 100%. It can be concluded that NIRS was an accurate method of diagnosis of pregnancy at Days 18 and 25 after AI in ewes.     

Effect of feeding yeast culture on reproduction and lameness in dairy cows under heat stress

Multiparous Holstein cows (n=717) from two dairy farms were blocked at calving by parity and previous lactation milk yield and, within each block, randomly assigned to one of two treatments: a diet containing no yeast culture (Control; n=359) or 30 g/d of a culture of Saccharomyces cerevisiae (YC; n=358) from 20 to 140 d postpartum. Only cows calving during months of heat stress, May-August were enrolled. Lameness score (1-5 scale) was evaluated at study enrollment and again at 100 d postpartum. The body condition score (BCS, 1-5 scale) was evaluated at calving, 28, 58 and 140 d postpartum. Cows received two injections of PGF(2alpha) at 37 and 51 d postpartum, and those observed in estrus were inseminated. Cows not in estrus were enrolled in a timed AI protocol at 65 d postpartum and inseminated at 75 d postpartum. Ovaries were examined by ultrasonography at 37 and 51 d postpartum to determine whether estrous cycling had been initiated by the presence of a corpus lutem (CL) in at least one of the two examinations. Pregnancy was diagnosed at 31, 38 and 66 d after the first AI and at 38 and 66 d after the second and third AI. Diet did not affect time of onset of estrous cycles postpartum, and 8.2% of the cows were anovular. Detection of estrus in the 7d after the second injection of PGF(2alpha) was similar for control and YC. For control and YC, conception rates 38 d after AI at first (30.8% and 31.4%), second (39.3% and 35.1%) and third (25.8% and 30.6%) inseminations, and pregnancy losses did not differ, which resulted in similar median days to pregnancy and proportion of pregnant cows at 140 d postpartum. Yeast culture did not affect incidence of lameness, but tended to reduce lameness score. Lame cows and anovular cows had lesser conception rates at first AI, and extended interval from calving to conception. A THI of 71 was identified as the critical point in which fertility was reduced in lactating dairy cows, although the sensitivity and specificity were minimal. Cows exposed to a THI>71 on the day of first AI had a 33% reduction in the rate of pregnancy resulting in extended interval to pregnancy. Feeding a yeast culture of S. cerevisiae had minor effects on lameness score, but no impact on reproduction of multiparous cows under heat stress.     

Effects of abnormal ovarian cycles during pre-service period postpartum on subsequent reproductive performance of high-producing Holstein cows

The primary objective of this study was to investigate the effects of abnormal ovarian cycles during the pre-service postpartum period on subsequent reproductive performance of high-producing Holstein cows. The study was conducted in a commercial dairy farm with approximately 150 lactating cows, in a subtropical region of Japan. Animals were kept in free-stall barn, and fed a total mixed ration. Cows that calved from June 2001 to July 2002 were included in the study. Milk samples were collected twice weekly from 2 to 11 weeks postpartum, and progesterone concentrations in skim milk were determined by ELISA. After a voluntary waiting period of 40 days, cows detected in estrus were bred by artificial insemination (AI). Pregnancy was confirmed by palpation per rectum 40-70 days after AI. Out of a total of 91 cases, 39 (42.9%) had normal ovarian cycles (ovulation within 45 days after calving, followed by normal ovarian cycles), 32 (35.2%) had prolonged luteal phase (i.e. luteal activity for >20 days), and 12 (13.2%) had anovulation or delayed first ovulation (i.e. first ovulation did not occur until >45 days after calving). The remaining (8.8%) had other types of abnormalities. When compared with cows with a normal ovarian cycle, prolonged luteal phase cows had a lower 100 days AI submission rate, conception rate and pregnancy rate (84.2% versus 56.3%; P<0.05, 50% versus 16.7%; P<0.05 and 42.1% versus 9.4%; P<0.01, respectively), and longer intervals to first AI and to conception ( 67+/-6 days versus 98+/-7 days and 95+/-9 days versus 136+/-11 days; P<0.01 for each). Similarly, when compared with cows with normal ovarian cycles, those with anovulation had lower 100 days conception rate and pregnancy rate (50% versus 0%; P<0.05 and 42.1% versus 0%; P<0.01, respectively), and longer intervals to first AI and to conception ( 67+/-6 days versus 93+/-12 days; P<0.05 and 95+/-9 days versus 155+/-14 days; P<0.01, respectively). Survival analysis of the data for calving to conception interval showed that cows with prolonged luteal phase or anovulation were getting pregnant at a slower rate, and took longer to get pregnant than the cows with normal resumption of ovarian cyclicity postpartum. In conclusion, abnormal ovarian cycles during the pre-service period postpartum adversely affected reproductive performance, including AI submission rate, pregnancy rate, interval to first AI, and calving to conception interval in high-producing Holstein cows.     

Endocrinology of pregnancy and early pregnancy detection by reproductive hormones in reindeer (Rangifer tarandus tarandus)

The endocrinology was studied throughout pregnancy in reindeer (Rangifer tarandus tarandus) located in Oulu, Finland (65 degrees N, 25 degrees E) with 13 captive, semi domestic adult females. Blood samples were analyzed for plasma progesterone (P4), estradiol (E2) and estrone sulphate (E1SO4), 15-ketodihydro-PGF2alpha (PG-metabolite) and pregnancy associated glycoproteins (PAG). The mean plasma P4 concentration peaked twice during gestation: at around 24 and three weeks prior to calving. In pregnant females the plasma PAG concentration increased over basal concentrations 21-30 days after the estimated day of conception and peaked at the time of calving. The concentrations of E2 and E1SO4 remained low until 60 days before calving when a rapid increase was found for both hormones. The mean plasma concentration of PG-metabolite increased throughout pregnancy to a maximum at parturition. The estimated mean (range) gestation length was 216 (212-220) days. Judged from measures on reproductive organs collected from 86 free-ranging, semi-domestic female reindeer of unknown age presented for slaughter at Roros, Norway (63 degrees N, 11 degrees E) in the second week of December 1999, it was concluded that the breeding season lasted from early September until the end of November. The results also showed that plasma PAG concentration could provide a tool for detection of pregnancy in reindeer.     

Effect of early postpartum PGF2α treatment on reproductive performance in dairy cows with calving and puerperal traits

The objective of this study was to evaluate the effects of early postpartum PGF two alpha treatment on reproductive performance in dairy cows with calving and puerperal traits. A total of 363 Holstein cows (128 primiparous and 235 multiparous) were selected based on the presence of at least one of calving and puerperal traits (dystocia, retained placenta, twin, abortion, and postpartum uterine infections) and were assigned to two groups (treatment and control) irrespective of presence or absence of luteal tissue. Cows in the treatment group were treated twice with 25 mg dinoprost 8 h apart on day 20 postpartum, and for the control group saline placebo was administered. As it was speculated that the timing of a second dose would mimic the release of endogenous PGF2α from the uterus, our hypothesis was that two doses of PGF2α 8 h apart may increase the duration of elevated plasma prostaglandin F2α metabolite concentration in these cows. Recorded reproductive variables included days to first estrus, days to first AI, first service conception rate, pregnancy by 150 days in milk, service per conception, open days, and the percentage of repeat breeder animals. The data were analyzed using SPSS (Version 15) (IBM North America, New York, NY, USA) and Minitab (Version 14) (Minitab, State College, PA, USA). Although early postpartum PGF2α treatment had no effect on days to first estrus (36.7 days vs. 34.9 days, P = 0.056) and days to first AI (70.5 days vs. 72.2 days, P = 0.537), it increased first service conception rate (47.1% vs. 27.6%, P < 0.001); and this was more remarkable in primiparous cows (64.7% vs. 25%, P < 0.001). PGF2α treatment reduced the mean service per conception (1.92 vs. 2.72, P < 0.001) and the mean open days (112 days vs. 144 days, P < 0.001), and increased pregnancy by 150 days in milk (DIM) (80% vs. 66%, P = 0.004). The prevalence of repeat breeder syndrome in cows with calving and puerperal traits was reduced by PGF2α treatment (10% vs. 29.8%, P < 0.001). In conclusion, treatment of cows with calving and puerperal traits twice with a luteolytic dose of PGF2α 8 h apart on Day 20 postpartum improved reproductive performance and reduced the prevalence of repeat breeder syndrome.     

Comparison of reproductive performance by artificial insemination versus natural service sires in California dairies

This study compared the calving to conception intervals for cows in AI pens with cows exposed to natural service sires, controlling for milk production, mastitis occurrence, parity and calving month effects. Records from 10 western United States dairy herds (mean herd size = 2058 cows) were evaluated retrospectively over an 18-month period. Eight bull breeding analysis cohorts were created (the first cohort 0-50 days in milk and the remaining cohorts at 25 days in milk intervals through 226 days). The cohorts contained non-pregnant cows that were first moved into bullpens during the described cohort period. Equal numbers of non-pregnant cows only exposed to AI during the cohort period were randomly selected from the pool of eligible non-pregnant cows. An AI cow was used only once in the data analysis, but was included in a bull breeding cohort at a later date if she remained non-pregnant and was transferred to a bullpen. Univariate and multivariate survival analysis was used to compare the calving to conception intervals. Cows in AI groups had higher hazard rates for pregnancy across all cohorts. Parity and milk production were significantly associated with risk for pregnancy. In herds that practice a mixture of AI and bull breeding, overall herd reproductive performance might be improved by allowing cows more opportunities at AI prior to moving them into clean-up bullpens.     

Relationship of pre-ovulatory follicle size, estradiol concentrations and season to pregnancy outcome in dairy cows

This study was carried out to evaluate effects of pre-ovulatory follicle size, plasma concentrations of estradiol and progesterone, and season on pregnancy outcomes in dairy cows. Holstein cows (n = 144) were synchronized and inseminated (Ovsynch/TAI protocol) in two distinct periods (cold versus warm season). Blood samples were collected daily from AI (day 0) to day 8 and on days 15, 22, 29, 36 and 64 to measure progesterone and estradiol. Pregnancy diagnosis was performed at days 29, 43 and 64. The pre-ovulatory follicle size was larger and the plasma estradiol concentrations on the day of AI were greater in animals that became pregnant. Plasma progesterone concentrations diverged and became greater after day 5 post-AI, in cows diagnosed pregnant, as compared to non-pregnant cows. The overall pregnancy rate (33%) or late embryonic/early fetal losses (23%) did not differ between seasons, but plasma estradiol concentrations on the day of AI and plasma concentrations of progesterone in pregnant cows were lower in the warm season. Reduced CL function, measured as plasma progesterone concentrations, from days 22 or 29 post-AI onward for cold and warm season, respectively, was associated with subsequent late embryonic/early fetal mortality. Overall, pregnancy was related to diameter of the pre-ovulatory follicle and plasma E2 on the day of AI, but embryonic/fetal losses were not. Season did not affect these outcomes, even though it influenced luteal function after AI.     

Early pregnancy diagnosis in sheep by progesterone and pregnancy-associated glycoprotein tests

The aim of this study was to compare the accuracy of the progesterone (P4) and pregnancy associated glycoprotein (PAG) tests for determination of early pregnancy in sheep. Estrus was synchronized in 182 Awassi x Merino ewes and blood samples were collected at Days 0 (day of the insemination), 18, 22, 29, 36, and 50 after artificial insemination (AI). Plasma P4 concentrations at Days 0 and 18 were determined by double antibody radioimmunoassay, while PAG concentrations at Days 22, 29, 36 and 50 were determined by a heterologous, double-antibody radioimmunoassay (RIA) using the bovine PAG 67 kDa subunit as tracer and standard and rabbit antiserum raised against a mixture of caprine 55 and 59 kDa PAG subunits as the first antibody. The discriminatory value for diagnosis of pregnancy by the P4 and the PAG-RIA tests was > or = 1 ng/ml. Based on lambing data, the accuracy for diagnosing pregnant (sensitivity) and non-pregnant ewes (specificity) and predictivity of both tests were calculated. The sensitivity, specificity, positive and negative predictive values for P4 and PAG tests were 100, 95.4, 81.6, and 100% at Day 18 (P4) and 93.5, 100, 100 and 98.7% at Day 22 (PAG), respectively. For diagnosis of non-pregnant ewes the PAG test had significantly higher specificity than the P4 test (P < 0.05). It is concluded that ovine pregnancy can be reliably diagnosed at Day 22 after AI by using a heterologous radioimmunoassay of PAG.     

Incorporation of a rapid pregnancy-associated glycoprotein ELISA into a CIDR-Ovsynch resynchronization program for a 28 day re-insemination interval

The objective was to compare two resynchronization programs; one that used a blood-based ELISA for pregnancy-associated glycoproteins (PAG) for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 28 d after first TAI, and another that used transrectal ultrasonography for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 35 d after first TAI. The PAG_resynch cows (n = 103) began CIDR-Ovsynch resynchronization on Day 18 after first TAI (Day 0). On Day 25, the CIDR was removed and pregnancy diagnosis with a PAG ELISA was performed. If a cow was not pregnant on Day 25, she was treated with PGF_2α, treated with GnRH 2 d later (Day 27), and TAI on Day 28. Control cows (n = 99) were observed for estrus until Day 25, when they began an identical CIDR-Ovsynch program with pregnancy diagnosis by transrectal ultrasonography on Day 32. If a cow was not pregnant on Day 32, then she was treated with PGF_2α, treated with GnRH 2 d later (Day 34), and TAI on Day 35. There was no difference in pregnancy per AI (P/AI) for either group at first or second insemination. For cows without pregnancy loss, the interval between first and second (P < 0.001) or second and third (P < 0.016) TAI was shorter for PAG_resynch cows compared with Control cows. The interval between first and second or second and third TAI was not different if pregnancy loss cows were included in the analysis. Plasma progesterone concentrations were similar at PGF_2α treatment, and plasma estradiol concentrations increased similarly after PGF_2α treatment for PAG_resynch and Control cows. In conclusion, the 28 d CIDR-Ovsynch resynchronization protocol was comparable to a 35 d CIDR-Ovsynch resynchronization protocol that also included estrus detection. Shortened resynchronization protocols that do not require estrus detection may improve reproductive efficiency in dairy cattle.     

Neospora caninum and coxiella burnetii seropositivity are related to endocrine pattern changes during gestation in lactating dairy cows

Q fever is a zoonotic infection caused by Coxiella burnetii that is endemic worldwide. Domestic ruminants are a source of infection for humans. Given the suggestion that the bacterium recrudesces during pregnancy in cattle, this study was designed to determine whether C. burnetii infection affects hormonal patterns, such as progesterone, cortisol, pregnancy associated glycoproteins (PAG), and prolactin during gestation in lactating cows. Possible interactions with Neospora caninum were also explored. The study was performed on 58 gestating non-aborting cows. Blood samples for hormone determinations were collected on Days 40, 90, 120, 150, 180, and 210 of gestation. For antibody determinations, blood was collected at day 40 postinsemination and postpartum. By GLM repeated measures analysis of variance, we established the effects of production and reproductive variables as well as Coxiella and Neospora seropositivity related to changes on cortisol, PAG, progesterone, and prolactin levels. Coxiella antibody levels were significantly related to cortisol, PAG, and plasma progesterone concentrations, whereas Neospora seropositivity was linked to plasma progesterone concentrations. The interaction between Coxiella and Neospora seropositivity was correlated with cortisol and plasma progesterone levels, whereas the interaction seropositivity against C. burnetii-plasma cortisol concentration was related to plasma PAG levels. Finally, an effect of lactation number only was observed on plasma prolactin. Our findings suggest that both the N. caninum and C. burnetii infection or the presence of both modify endocrine patterns throughout gestation. Cows seropositive to both, Neospora and Coxiella, showed higher plasma progesterone levels than the remaining animals examined. Seropositivity to C. burnetii was associated with placental damage and diminishing PAG levels throughout the second half of gestation, along with increased plasma cortisol levels on Day 180 of gestation.     

Ovarian and endocrine responses and reproductive performance following GnRH treatment in early postpartum dairy cows

At calving forty-eight Holstein and Guernsey cows were assigned according to age and breed to one of six postpartum periods (1 or 2, 3 or 4, 5 or 6, 7 or 8, 12 or 13 and 18 or 19 days postpartum). Thirty-six of the cows (6 cows per postpartum period) received a single intramuscular injection of 100 μg GnRH. The other twelve cows (2 cows per postpartum period) served as controls and received a single intramuscular injection of the carrier vehicle for GnRH.Four of 36 cows administered GnRH and three of the 12 control cows ovulated by the day following treatment. Four of the cows were 12 or 13 days postpartum (1 control and 3 GnRH treated) and three were 18 or 19 days postpartum (2 controls and 1 GnRH treated). Six of the seven cows that ovulated the day following treatment had a follicle > 1.0 cm the day prior to treatment. Follicular growth was detected in the earlier postpartum periods but ovulation the following day was not detected for either control or GnRH treated cows. Following estrus or silent estrus, plasma progesterone concentrations increased to about 4 ng/ml on day 13. However, in cows ovulating the day following GnRH treatment, plasma progesterone declined from about 3 ng/ml on day 9 to approximately 1 ng/ml on day 13 postestrus. In addition, LH in plasma was higher (P < .01) ? through 13 days following estrus or silent estrus in cows ovulating the day after GnRH treatment in comparison to cows during the first or subsequent postpartum estrous cycles.In summary, in addition to days postpartum other factors including follicular development and maturity are probably involved in GnRH induced ovulation.     

Pregnancy-specific protein B and progesterone concentrations in French Alpine goats throughout gestation.

The 34 French Alpine dairy goats originated from a single flock and were artificially inseminated 44 h after synchronization of oestrus. They were bled daily at the jugular vein from 15 to 27 days after AI. An early pregnancy diagnosis by RIA of progesterone concentration was performed 21 days after AI. In pregnant goats (greater than 1.5 ng progesterone/ml) daily sampling was extended until 30 days after AI and, from those, 9 were bled every 2 weeks until the end of pregnancy and at 50 and 63 days post partum. Pregnancy-specific protein B (PSPB) was also assayed. The kidding rate was 67.6% (23/34). PSPB concentrations (ng/ml) in pregnant goats were significantly different from those of non-pregnant goats at 24 days after AI (0.82 +/- 0.18 vs 1.78 +/- 0.19; mean +/- s.e.m.) and rose to 40 ng/ml at the end of pregnancy. From Day 25 and throughout gestation, females with 2 fetuses had higher PSPB concentrations than did those with a single fetus (P less than 0.05). In the 2 goats exhibiting late embryonic mortality according to progesterone concentrations, one had a PSPB profile very similar to those of pregnant goats until 30 days while the other did not show any elevation of PSPB concentration. It is concluded that PSPB profiles in goats are similar to those found in cows throughout pregnancy and that PSPB RIA may be useful for pregnancy diagnosis or diagnosis of late embryonic mortality.     

Reproductive performance of postpartum dairy cows under a highly intervenient breeding program involving timed insemination and combinations of GnRH, prostaglandin F2alpha and human chorionic gonadotropin

Lactating Holstein cows (n=288) were grouped as pairs at parturition and randomly assigned to two treatments (control, C vs intervenient treatment, T). The reproductive management of the Group C cows (n=130) consisted of the intramuscular administration of 500 microg PGF2alpha analogue (PG) on Days 28 and 63 postpartum and breeding on the basis of estrus signs with the a.m.-p.m. rule after Day 63. Cows that were not bred by 77 d postpartum received another injection of PG and were bred at estrus or 84 h after PG treatment. Pregnancy diagnoses were perfomed by palpation of the uterus per rectum 42 to 48 d after AI. Cows in the T group (n=139) received intramuscular injections of 100 microg GnRH 14 d and PG 28 d after calving. On Day 56 postpartum, cows were given a second dose of GnRH followed by PG on Day 63 postpartum and a third GnRH injection 48 h after PG (OvSynch). Cows were inseminated at a fixed time (22+/-1 h) after GnRH. Five days after the fixed-time insemination cows were given 1500 IU hCG i.m.. Group C and T cows that returned to service or were diagnosed as non-pregnant continued to receive PG at intervals of 14 d with breeding at estrus or 84 h after the second PGF2alpha dose. A sustained increase in milk progesterone concentration was observed in 59.0% of T cows after GnRH administration on Day 14. A similar rise in milk progesterone concentrations was observed in 53.8% of C cows. The PG on Day 28 induced luteolysis more in Group T cows (53.2%) than in Group C cows (36.9%). The PG on Day 63 reduced milk progesterone concentrations to basal levels in 50.7% of T and 49.2% of Group C animals. The first service pregnancy rates (T, 40.3% vs C, 36.2%) and the overall pregnancy rates (all services, T, 83.5% vs C, 86.9%) were not different between the two groups. The two treatments did not differ in the interval from first service to pregnancy, calving to pregnancy or in calving interval, number of services per pregnancy or culling rates.     

Reproductive performance of repeat breeders in dairy herds

The objectives were to characterize repeat breeding in dairy cows, including reproductive performance and risk factors. Data from 613 Holstein Friesian cows in nine dairy herds across Japan were enrolled. A repeat breeder was defined as a cow that did not become pregnant after three inseminations, despite no clinically detectable reproductive disorders. In contrast, cows that became pregnant within three inseminations were considered to have normal fertility. Of the 613 cows, 87.3% eventually became pregnant after repeated AI (maximum calving to conception interval was 435 d). Mean (±SEM) first AI conception rate, days in milk at first AI, calving to conception interval and service per conception were 38.3%, 82 ± 2 d, 125 ± 3 d, and 2.0 ± 0.1 times, respectively. Normal fertility cows (n = 479) required only 114 ± 3 d to conceive and 1.7 ± 0.1 inseminations per pregnancy, whereas repeat breeders (n = 86) required significantly more days to conceive (211 ± 10) and more inseminations per pregnancy (4.7 ± 0.2). Based on survival analysis, it took 94 d after calving for 50% of normal fertility cows to become pregnant, compared to 155 d for repeat breeders. For repeat breeders, 31.4, 50.0, and 58.1% became pregnant within 210, 300, and 435 d after calving, respectively. The risk factors for repeat breeding were parity (relative risk [RR] = 0.809; P = 0.058), resumption of postpartum ovarian cycles (RR = 1.928; P = 0.009), and days in milk at first AI (RR = 0.991; P = 0.039). In conclusion, repeat breeder dairy cows had very poor reproductive performance. Lower parity, abnormal resumption of postpartum ovarian cycles, and shorter days in milk at first AI were risk factors for repeat breeding.     

Alteration in some pro and anti-inflammatory cytokines associated with complete and incomplete gestation cycle of cows

The maternal immune response during pregnancy is regulated by a complex array of pro and anti-inflammatory cytokines which provide optimum conditions for the embryo implantation and survival. The possible role of pro and anti-inflammatory cytokines during the complete gestation cycle of ruminants and the difference in their circadian rhythmicity between successful and unsuccessful pregnancies is still unknown. To study this, blood samples were collected from three groups of cows, pregnant (P), non-pregnant (NP) and aborted (ABORT) cows starting from the day of Artificial Insemination (AI) till calving in P cows and till stages of non pregnancy in other cows. Various pro and anti-inflammatory cytokines were estimated by bovine-specific ELISA test and compared. Successful pregnancies had lower levels of pro-inflammatory cytokines (IL-2, IL-6 and IL-8) but higher anti-inflammatory cytokine (IL-10) mainly during implantation. Significant (p < 0.05) increase in pro-inflammatory cytokines and low IL-10 levels was noticed at abortion and at calving. This study indicates that temporal and spatial aspects of reducing the release of various pro-inflammatory cytokines and maintaining high anti-inflammatory cytokines during pregnancy are essentially required for maintenance of pregnancy. Any disturbance in the cytokine equilibrium may lead to persistent inflammation and pregnancy failure.     

Plasma estrone sulphate (E1S) and estradiol-17beta (E2beta) profiles during pregnancy and their relationship with the relaxation of sacrosciatic ligament, and prediction of calving time in Holstein-Friesian cattle

The objectives of this study were to investigate the plasma E(1)S and E(2)beta profiles during pregnancy and their relationship with the relaxation of sacrosciatic ligament in Holstein-Friesian cattle (n=37) and then to predict the calving time on the basis of E(1)S and E(2)beta profiles and relaxation of the ligament. Blood samples were collected at 4 weeks intervals from days 100 to 190, at 2 weeks intervals from days 190 to 250, every week from days 250 to 270 and thereafter every day from day 270 of gestation until the day after calving. The relaxation in the ligament was measured by using two scales as a distance at a schedule similar to blood sampling plus 5 days postpartum. One scale was kept firm exactly parallel to the ligament between the sacrum and the tuber ischii and other scale was erected perpendicularly to the first scale with the bottom just touching the ligament and the depth was measured in the second scale from the point where it touched the ligament to the point where it touched the first scale. Plasma samples were analyzed for E(1)S and E(2)beta by enzyme immunoassay. E(1)S concentration was low at day 100 (0.8+/-0.3 ng/ml), then increased progressively and drastically to reach the level of 28.4+/-3.6 ng/ml on the day before calving and declined significantly (p<0.05) at 9.5+/-3.1 ng/ml within 1 day postpartum. There was a gradual increase in concentration of E(2)beta from day 100 of gestation (0.1+/-0 ng/ml) until day 4 prepartum (0.6+/-0 ng/ml). Thereafter, it increased drastically and reached the peak level of 1.0+/-0.1 ng/ml (p<0.05) on the day before calving and declined markedly at 0.4+/-0.1 ng/ml within 1 day postpartum (p<0.05). Corresponding to E(1)S and E(2)beta concentrations, a gradual increase in the relaxation of the ligament was observed from day 100 of gestation (8+/-1mm) until day 2 prepartum (24+/-2mm). Thereafter, it showed a significant increase (p<0.05) within 1 day before calving (31+/-2mm) and almost no difference between day 1 prepartum and day 1 postpartum. A marked decrease (p<0.05) was observed thereafter until day 3 postpartum (10+/-2mm) and no significant change between days 3 and 4 as well as 4 and 5 postpartum. The increment of E(2)beta by >or=0.20 ng/ml from the preceding day concentration was 85.2% accurate for predicting calving within 24h in many of the cows (23 of 37) in the herd. The increment in ligament relaxation measurement by >or=5mm from the preceding day measurement was the most efficacious to predict calving within 24h with the highest accuracy (93.9%) in high proportions of cows (31 of 37) in the herd. In conclusion, plasma E(1)S and E(2)beta concentrations and relaxation of sacrosciatic ligament increased gradually as gestation advanced and reached the peak level on the day before calving. The relaxation in the ligament corresponded well to plasma E(2)beta concentrations. Prediction of calving was possible by E(2)beta profile and relaxation in the ligament but not by E(1)S profile. The increment in ligament measurement by >or=5mm from the preceding day measurement was the most useful and accurate in predicting calving within 24h. It is economical and easily applicable in the field condition.     

Influence of bull biostimulation, season and parity on resumption of ovarian activity of zebu (Bos indicus) cattle following parturition

A total of 135 postpartum suckled zebu cows were assigned randomly at calving to two treatments: cows exposed to vasectomised bulls (Mature Bull Exposure, MBE) and cows not exposed to bulls (No Bull Exposure, NBE) for a period of 150 days postpartum. This study was conducted to determine the influence of bull biostimulation, season and parity on postpartum reproductive performance of Bos indicus cattle. The trials were conducted in two seasons: cows calving in the dry season and cows calving in the rainy season. Cows with an increase in serum progesterone (P(4)) concentration of >1 ng/ml from the weekly blood samples were used to analyse the number of days from calving to the time of resumption of ovarian activity. The introduction of vasectomised bulls shortened the postpartum anoestrus in cattle following calving. Mean interval from calving to resumption of ovarian activity for the MBE cows was 71.7 days, which was significantly earlier following calving than the NBE cows with a duration of 77.8 days. By 60 to 80 days postpartum, the proportion of cows at resumption of postpartum activity for MBE cows was greater than for the NBE cows.Mean interval from calving to resumption of ovarian activity for cows that calved in the dry season was 71.3 days, which was significantly earlier than for cows that calved in the rainy season (78.6 days). At 60 days postpartum, the proportion of cows at resumption of postpartum ovarin activity for cows that calved in the dry season was greater than the cows that calved in the rainy season. Mean interval from parturition to resumption of ovarian activity for cows with three to five calvings was 65.1 days, which was significantly earlier than the value of 71. 2 days for cows with one to two calvings. By 60 to 80 days postpartum, the proportion of cows at onset of ovarian activity for cows with three to five calvings was greater than those cows with one to two calvings. Cows that calved in the dry season completed uterine involution by 24.4 days, which was significantly shorter than the duration of 26.5 days for cows that calved in the rainy season. Cows with three to five calvings completed uterine involution earlier than those with one to two calvings. It is concluded that bull-cow biostimulation influences reproductive activity in the cow possibly through olfactory cues (pheromones).