Aly Karen

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.

Estimation of gestational age in Egyptian native goats by ultrasonographic fetometry

The main aim of the present study was to estimate the gestational age of Egyptian goats by B-mode ultrasound measurement of embryonic or fetal parts throughout pregnancy. Trans-rectal (TR) ultrasonography (7 MHz) was carried out on 15 pregnant Egyptian does at Day 10 post mating on alternate days until Day 25 and then once at 3-5-day intervals until Day 50. Trans-abdominal (TA) ultrasonography (3.5-5 MHz) was carried out on the same animals from Days 25 to 130 at 3-5-day intervals. After imaging the embryo or the fetus, the following parameters were measured: length of the embryo or fetus (CRL), heart rate (FHR), biparital diameter (BPD), trunk diameter (TD), placentome size (PS), umbilical cord diameter (UCD) and femur length (FL). The average of days at which the embryonic vesicle was first determined by TR and TA ultrasonography was 16.98+/-1.97 and 27.87+/-3.48, respectively. The embryo proper with a beating heart was first determined by TR and TA ultrasonography at an average of 22.36+/-2.66 and 30.36+/-4.75 days, respectively. All the fetal measures were significantly (P<0.0001) correlated with the gestational age. With the exception of fetal heart rate (R(2)=0.551), all the measured fetal structures were highly correlated (R(2)> or =90) with the gestational age. In conclusion, the age of embryo or fetus in Egyptian does can be estimated by ultrasound measuring the crown rump length, biparital diameter, trunk diameter, placentome size, umbilical cord diameter and femur length.

Efficacy of Ovsynch protocol in cyclic and acyclic Egyptian buffaloes in summer.

The aim of the present study was to evaluate the efficacy of an Ovsynch protocol in the cyclic and non-cyclic Egyptian buffaloes in the summer. The present study was carried out on 21 Egyptian pluriparous buffalo cows (11 cyclic and 10 acyclic) and 8 heifers (5 cyclic and 3 acyclic). All animals were administered 100 microg GnRH i.m. at Day 0, 500 microg of PGF2 proportional, variant i.m. at Day 7 and a second dose (100 microg) of GnRH i.m. at Day 9. Ovarian structures of the animals were recorded by means of transrectal ultrasonography (6-8 MHz linear-array transducer) daily from Day 0 (before treatment) to Day 9 (the second GnRH administration) and thereafter, at 12-h interval till ovulation or for a maximum of 48 h. After ultrasound examination, a blood sample was taken daily from each animal for progesterone analysis. The buffalo cows were inseminated at 16-20 h after 2nd GnRH administration. After the first administration of GnRH, 46% (5 of 11) of cyclic and 50% (5 of 10) of acyclic cows and 40% (2 of 5) of cyclic and 33% (1 of 3) of acyclic heifers responded by ovulation or luteinization of the follicles. The mean diameters (+/-S.E.M.) of the follicles in the responding cows and heifers were significantly higher than those in non-responding animals (9.7 +/- 0.4 mm vs. 6.7 +/- 0.6 mm, P < 0.0001; 11.1 +/- 1.5 mm vs. 7.3 +/- 0.7 mm, P < 0.05, respectively). After the second administration of GnRH, 81% (9 of 11) of cyclic and 60% (6 of 10) of acyclic cows and 80% (4 of 5) of cyclic and 33% (1 of 3) of acyclic heifers ovulated, respectively. Ovulations occurred earlier (P = 0.059) and over a wider range of time in the non-cyclic cows (26 +/- 4.8 h; range: 12-36 h) and in a heifer (12 h,) than in the cyclic cows (34.7 +/- 1.3 h; range 24-36 h) and heifers (33 +/- 3 h; range: 24-36 h). The conception rate was 18% (2 of 11) and 0% (0 of 10) in cyclic and non-cyclic cows, respectively. In conclusion, the Ovsynch protocol could be used effectively for synchronization of ovulation in cyclic cows and heifers in summer.

Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle.

The present study aimed to compare the accuracy of a commercial PAG-ELISA test (Bovine Preg Test 29) and bovine pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) for diagnosing pregnancy at Day 28 after insemination in dairy cows. Transrectal ultrasonography (TRUS) was performed in 100 Holstein-Friesian cows at Day 28 after artificial insemination (AI; Day 0) to diagnose pregnancy. After TRUS examination, blood sample was collected from the coccygeal vessels of each cow to measure the concentrations of bPAGs by PAG-RIA test and Bovine Preg Test 29. Milk samples were collected at Days 0, 21 and 28 for measurement of progesterone (P4) by ELISA test. The cows were re-examined by TRUS at Day 42 to confirm the pregnancy diagnoses. The actual gold standard was based on TRUS outcomes at Day 28 that agreed with the outcomes of PAG-RIA test or PAG-ELISA test. If the outcomes of TRUS at Day 28 and PAG-RIA test and PAG-ELISA test did not agree, the gold standard was based on the outcome of TRUS at Day 42. Out of 100 inseminated cows, 41 were confirmed pregnant at Day 28 after AI. Based on the actual gold standard, the sensitivity of TRUS, PAG-ELISA and PAG-RIA tests for diagnosing pregnant cows at Day 28 were 92.7%, 90.2% and 100%, while the specificity of the three tests for diagnosing non-pregnant cows were 91.5%, 98.3% and 94.4%, respectively. The overall accuracy of the three tests were 92%, 95% and 97%, respectively. The degree of agreement (Kappa±S.E.) between PAG-RIA and PAG-ELISA test was 0.90 ±0.04. The degrees of agreement between PAG-RIA and PAG-ELISA and TRUS at Day 28 were 0.80±0.05 and 0.76±0.06, respectively. In conclusion, the commercial PAG-ELISA test is a highly accurate method for diagnosing early pregnancy in dairy cows on Day 28 after AI and may be used as an alternative method to the TRUS and the PAG-RIA test.

Effect of restraint stress on plasma concentrations of cortisol, progesterone and pregnancy associated-glycoprotein-1 in pregnant heifers during late embryonic development.

The aim of the present study was to evaluate the effect of restraint stress, which is commonly practised in the field, on plasma concentrations of cortisol, progesterone (P4) and bovine pregnancy-associated glycoprotein-1 (boPAG-1) in pregnant heifers between Days 30 to 40 of gestation. Twelve Holstein-Friesian heifers between Days 30 (Day 0 of experiment) and 40 (Day 10 of experiment) of pregnancy in a Hungarian dairy farm were used in the present study. The heifers were exposed to an acute stressor consisting of immobilisation (restraint stress) in a crush for 2 h (Group 1, n = 6) on Day 2 (Hour 48) and for 2 × 2 h (Group 2, n = 6) on Days 2 and 3 (Hour 72) of the experiment. Transrectal ultrasonography (7.5 MHz linear-array rectal transducer) was performed daily from Day 0 to Day 10 of the experiment to detect embryonic heartbeat or the fate of the conceptus. Blood samples were withdrawn before each ultrasonographic examination. Additional blood samples were withdrawn by 1 and 2 h (at Hours 49 and 50 in Groups 1 and 2 and Hours 73 and 74 in Group 2) of the onset of applying the stressor. Plasma cortisol, P4 and boPAG-1 concentrations were measured by radioimmunoassay. Acute restraint stress significantly (P < 0.001) increased the plasma cortisol level in pregnant heifers at 1 h of the exposure to the stressor at Days 2 (48 h) and 3 (72 h) of the experiment. On the other hand, the restraint stress did not affect the concentration of P4 and boPAG-1 concentrations in both groups. In conclusion, restraint stress for 2 h during early pregnancy in heifers increased blood cortisol, but it did not affect the concentrations of P4 and boPAG-1 between Days 30 to 40 of gestation.

Effect of pretreatment with insulin on the response of buffaloes with inactive ovaries to gonadotrophin-releasing hormone agonist treatment in summer

The aim of the present study was to evaluate the effect of a pretreatment with insulin on the response of buffalo cows with inactive ovaries to gonadotrophin-releasing hormone agonist (GnRHa) treatment during hot summer months (July and August). Thirty-six Egyptian buffalo cows with inactive ovaries were randomly allocated into three groups: (1) group treated with GnRHa (G1, n = 16) in which each buffalo received an intramuscular injection of 250 mg of GnRHa (Day 0; gonadorelin, Fertagyl); (2) group treated with insulin before the GnRHa injection (G2, n = 8) in which each buffalo received a subcutaneous injection of biphasic insulin at a dose of 0.25 IU kg(-1) bodyweight once daily starting at Day -3 for 3 consecutive days, followed by an intramuscular injection of 250 mg of GnRHa on Day 0; and (3) the control group (G3, n = 12) in which each buffalo cow received an intramuscular injection of 2.5 mL of sterile saline on Day 0. The ovaries of all animals were examined by trans-rectal ultrasonography (5 MHz) on Days -7, -3 and 0 and continued thereafter at 4-day intervals until oestrus or the end of the experiment. On the same days that ultrasound examinations were performed blood samples were collected to measure the progesterone concentrations. Administration of insulin for 3 days before GnRHa injection (G2) significantly (P < 0.05) increased the diameter of the largest follicle from 6.85 +/- 0.64 to 12.4 +/- 0.88 mm. There was a significant (P < 0.01) increase in the oestrous induction rate in G2 compared with both G1 and G3 during the first 12 days after the treatment interval. It is concluded that pretreatment with insulin for 3 days before GnRHa injection increases the diameter of the dominant follicle and therefore the oestrous induction rate of acyclic buffaloes.

Relationship of progesterone, bovine pregnancy-associated glycoprotein-1 and nitric oxide with late embryonic and early fetal mortalities in dairy cows

The aim of the present study was to determine the relationship of progesterone (P4), bovine pregnancy-associated glycoprotein-1 (bPAG-1) and nitric oxide (NO) levels with late embryonic (LEM; day 28 to day 42) and early fetal mortalities (EFM; > day 42 to day 56) in dairy cows. Transrectal ultrasonography (6–8 MHz) was performed in 100 Holstein-Friesian cows at days 28, 42 and 56 after artificial insemination (AI; day 0) to diagnose pregnancy and to monitor the fate of the embryo. After ultrasound scanning of each cow, a milk sample was collected for assessment of P4 by an ELISA test and a blood sample was collected for assessment of bPAG-1, by using a double-antibody radioimmunoassay, and serum NO metabolites (nitrate + nitrite). Based on ultrasonographic examinations and bPAG-1-RIA, 41 of 100 inseminated cows were confirmed pregnant at day 28 after AI. Nine cows suffered of LEM, and 6 cows suffered of EFM and the overall pregnancy loss rate was 36.6% (15/41) between days 28 and 56 of pregnancy. By logistic regression analysis, there were no significant relationships between the level of P4 and bPAG-1 at day 28 after AI and the occurrence of LEM and EFM. Also, there were no significant relationships between the levels of P4 and bPAG-1 at day 42 and the occurrence of EFM. On the other hand, a significant relationship (P<0.05) was found between NO level at day 28 and the occurrence of LEM. In conclusion, measurement of the serum NO concentration at day 28 of pregnancy might help to predict the outcome of pregnancy by day 42 in dairy cows but further studies are needed to confirm this.

Accuracy of B-mode ultrasonography for diagnosing pregnancy and determination of fetal numbers in different breeds of goats

A total of 150 does of different breeds were used to evaluate the accuracy of transrectal (TRU) and transabdominal ultrasonography (TAU) for pregnancy diagnosis and fetal number estimation and to study the effect of breed and age of goats on the accuracy of ultrasonography. All does were scanned by TRU at days 17-22, 24-29 and 31-36 and by TAU at days 39-44 and 46-51 after breeding. The sensitivity (Se) of TRU for detecting pregnant does increased significantly (81.6% vs. 97.7%; P<0.0005) from days 17-22 to 24-29. There was a significant (P<0.05) effect of breed of goats on the specificity (Sp) of TAU for diagnosing non-pregnancy between days 39 and 51. In addition, there was a significant (P<0.01) effect of breed of goats on the Se and Sp of TRU for determination of does carrying multiples and singles, respectively, between days 24 and 36. The sensitivity of TAU for diagnosing pregnant does tended to be significantly (P=0.06) higher in young does (1.5-2 yr) than in older does (>2-9 yr) at days 39-44. The accuracy of TRU for determination of does carrying singles was significantly (P<0.05) higher in young does than that in older ones at days 24-29. In conclusions, TRU could be accurately used for early pregnancy diagnosis and fetal number estimation at days 24-29 and days 31-36, respectively, with accuracy similar to that of TAU at days 39-51. Furthermore, breed and age of does might influence the accuracy of ultrasonography for pregnancy diagnosis and fetal numbers estimation.

Effect of a single injection of gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) on testicular blood flow measured by color doppler ultrasonography in male Shiba goats

Although color Doppler ultrasonography has been used to evaluate testicular blood flow in many species, very little has been done in goat. Eight male Shiba goats were exposed to a single intramuscular injection of either gonadotropin-releasing hormone (GnRH group; 1 µg/kg BW) or human chorionic gonadotropin (hCG group; 25 IU/kg BW). Plasma testosterone (T), estradiol (E2) and inhibin (INH) were measured just before (0 hr) and at different intervals post injection by radioimmunoassay. Testis volume (TV) and Doppler indices, such as resistive index (RI) and pulsatility index (PI) of the supratesticular artery, were measured by B-mode and color Doppler ultrasonography, respectively. The results indicated an increase in testicular blood flow in both groups, as RI and PI decreased significantly (P<0.05), but this increase was significant higher and earlier in hCG group (1 hr) than in the GnRH group (2 hr). A high correlation was found for RI and PI with both T (RI, r= −0.862; PI, r= −0.707) and INH in the GnRH group (RI, r=0.661; PI, r=0.701). However, a significant (P<0.05) correlation was found between E2 and both RI (r= −0.610) and PI (r= −0.763) in hCG group. In addition, TV significantly increased and was highly correlated with RI in both groups (GnRH, r= −0.718; hCG, r= −0.779). In conclusion, hCG and GnRH may improve testicular blood flow and TV in Shiba goats.

Plasma urea nitrogen in relation to pregnancy rate in dairy sheep.

The aim of this field study was to investigate the relationship of plasma urea nitrogen (PUN) with the pregnancy rate in lactating Awassi × Merino ewes. One hundred and eighty-five Awassi × Merino ewes were used in the present study. Ewes were fed a diet containing 17.4% crude protein and were milked twice a day by the milking machine. The ewes were synchronized for estrus by insertion of intravaginal sponges containing 30 mg flurogestone acetate for 14 days. At the time of sponge removal each ewe was administered eCG (600 IU). All ewes were inseminated twice with fresh semen into the external os of the cervix at 48 and 56 h after sponge removal. The day of insemination was considered as Day 0 for calculating the gestational period. Blood samples were collected from each ewe at Days 0, 18 for measurement of PUN concentrations and at Day 22 after AI for measurement of pregnancy-associated glycoprotein (PAG) by radioimmunoassay (RIA). Thirty-eight ewes (20.5%) were confirmed pregnant by PAG-RIA test at Day 22 and by ultrasonography at Day 80. The mean (±S.D.) concentration of PUN in all ewes at Day 0 was 12.7±4.6 mmol/L. There were non-significant differences in the level of PUN between pregnant and non-pregnant ewes at Days 0 (12.2±4.2 mmol/L vs. 12.8±4.7 mmol/L, respectively) and 18 (9.6±2.9 mmol/L vs. 10.4±4.0 mmol/L, respectively) after AI. Mean PUN concentrations decreased significantly from Day 0 to Day 18 after AI in both pregnant and non-pregnant ewes. By using logistic regression analysis, there was no effect of PUN concentrations on the probability of pregnancy occurrence in the studied ewes (odds ratio: 0.97; 95% confidence interval: 0.9-1.05; P=0.45). In conclusion, there was no evidence of a relationship between PUN concentration and pregnancy rate for lactating Awassi × Merino ewes in the present study because of low pregnancy rate observed.