L. Zarco

Progesterone metabolism during storage of blood samples from Gyr cattle: Effects of anticoagulant, time and temperature of incubation

Ten Gyr cows with a functional corpus luteum were used to evaluate the effects of time and temperature of incubation of blood samples on progesterone (P4) concentrations detected in plasma or serum. From each cow, a blood sample was collected into a flask containing no anticoagulant, another into an heparinized flask and a third into a flask containing sodium fluoride. The blood from each flask was divided into 46 aliquots. One of them was centrifuged within 5 min of collection. The remaining 45 aliquots were divided into three groups and kept at three different temperatures: 4 degrees C, 17 degrees C, or 37 degrees C. For each anticoagulant, aliquots from every cow and incubation temperature were centrifuged every 30 min for 6 h, and then at 8, 12 and 24 h. Plasma or serum were separated immediately after centrifugation and were kept frozen at -20 degrees C until assayed for progesterone. The mean initial concentration of P4 in serum (8.3 ng/ml) significantly diminished (P<0.05) to 6.7 ng/ml after 5 h of incubation at 4 degrees C, 3 h at 17 degrees C, or 2 h at 37 degrees C. In plasma from heparinized blood the initial concentration (7.8 ng/ml) declined significantly after 6 h of incubation at 4 degrees C, 2 h at 17 degrees C, or 1 h at 37 degrees C. Sodium fluoride used as anticoagulant prevented the degradation of P4 since the initial concentration of P4 (6.7 ng/ml) never declined during incubation at either 4 degrees C or 37 degrees C; the only significant reduction occurred after 24 h of incubation at 17 degrees C.

PREMATURE LUTEAL REGRESSION IN GOATS SUPEROVULATED WITH PMSG: EFFECT OF hCG OR GnRH ADMINISTRATION DURING THE EARLY LUTEAL PHASE

Twenty-two goats were superovulated with PMSG; 84 h after the onset of estrus the goats were treated with saline solution (control group n = 7), hCG (hCG group, n = 7), or GnRH (GnRH group, n = 8). The ovaries of all the goats were laparoscopically examined 3 and 6 d after the onset of estrus. In each case the CL were counted and classified according to their appearance as normal-looking or as regressing. Blood samples for progesterone determination were collected every 12 h from Day 1 to Day 6. Premature luteal regression was considered to have occurred if progesterone concentrations declined to less than 1 ng/mL by Day 6. According to progesterone concentrations, 57.5, 0 and 37.5% of the goats underwent premature luteal regression in the control, hCG and GnRH groups, respectively. Progesterone concentrations were higher in the hCG group than in the other groups on Days 5 and 6 post estrus (P < 0.05). The control group was the only one in which there was a significant (P < 0.05) increase in the number of regressing CL between Day 3 (1.6 +/- 1.4) and Day 6 (7.3 +/- 1.4). It was also the only group in which there was a significant decrease in the number of normal-looking CL between Day 3 (12.6 +/- 2.1) and Day 6 (2.6 +/- 2.1). On Day 6 the animals treated with hCG had significantly more normal-looking CL (12.0 +/- 2.3) than those in the control group (2.6 +/- 2.1). The number of large follicles present on the ovaries on Day 6 post estrus had negative correlations with progesterone concentrations (P = 0.05) and with the number of normal-looking CL (P < 0.05). It is concluded that the administration of hCG 84 h after the onset of estrus prevents premature luteal regression in goats superovulated with PMSG.

Effect of short-term treatment with bovine somatotropin at estrus on conception rate and luteal function of repeat-breeding dairy cows.

We studied the effect of recombinant bovine somatotropin (rbST) at the time of estrus on progesterone concentrations and conception rates of repeat-breeding Holstein cows. We used repeat-breeding cows of varied parity (n = 510). All the animals were clinically healthy and had had at least three unsuccessful services before entering the study. After detection of estrus, the cows were randomly assigned to either a treated (n = 201) or a control (n = 309) group. The animals in the treated group were given rbST (500 mg s.c.) at the time of estrus and again 10 d later. Artificial insemination was performed 12 h after the first detection of estrus. In order to evaluate the effect of rbST on luteal function, blood samples were taken from 10 cows in each group every 3 d for 18 d, starting on the day of insemination (Day 0) to determine progesterone concentrations. Conception rates were significantly higher (P < 0.05) in the cows treated with rbST (29.3%) than in the control cows (16.9%). The effects of rbST were maximal in cows with 8 or more previous unsuccessful services and in cows with 2 to 4 calvings. Progesterone concentrations tended to be higher in nonpregnant cows that were treated with rbST than in those that were not treated. The difference between groups was significant (p < 0.05) on Day 18 after insemination. In pregnant cows there were no significant differences in progesterone concentrations between treated and nontreated animals at any time. Treatment with rbST at estrus improved the conception rate of repeat-breeding Holstein cows. This effect was associated with an increase in circulating progesterone concentrations on Day 18.

Estrus synchronization with melengestrol acetate in cyclic ewes. Insemination with fresh or frozen semen during the first or second estrus post treatment.

A total of 540 cyclic ewes were randomly allocated to 1 of 6 groups according to synchronization or not with melengestrol acetate (MGA), insemination with fresh or frozen semen, and insemination during the first or second estrus post treatment. The MGA was effective in synchronizing estrus, since the percentage of ewes showing estrus during the first 6 days after treatment was significantly higher (P<0.05) for treated (79.5%) than for nontreated ewes (33.5%); 74% of the treated ewes showed estrus during a 72-h period. Progesterone concentrations in plasma samples obtained at random from 34 treated ewes on Day 8 post estrus revealed that 94% of them ovulated and formed a functional CL. Synchronization was maintained during the second estrus post treatment, since 71.9% of the ewes showed the second estrus during a 72-h period. Treatment with 0.22 mg of MGA/head/d for 14 d had a detrimental effect on fertility when insemination was carried out during the first estrus post treatment. Delaying insemination until the second estrus post treatment caused a less marked reduction in conception rates. Thus, MGA can be a useful alternative for estrus synchronization of a large number of ewes. Artificial insemination can be delayed until the second estrus post treatment, improving fertility without loosing the advantages of estrus synchronization.

Incidence of delayed ovulation in Holstein heifers and its effects on fertility and early luteal function.

In the first of 3 experiments 134 first-service and 108 repeat-breeder Holstein heifers were palpated at 12-hour intervals starting 24 hours after insemination to compare the incidence of delayed ovulation in the 2 groups. Delayed ovulation was defined as failure to ovulate within the first 24 hours after insemination. Ovulation occurred within 24 hours post insemination in 92.1% of the animals and was delayed in 7.9% of the cases, with no differences between first-service and repeat-breeder heifers, indicating that the subfertility of the repeat-breeder animals was not due to delayed ovulation. The duration of the delay was at most 12 hours since all the animals had ovulated by 36 hours post insemination. Conception rate of the 19 animals with delayed ovulation (42.1%) was not different (P>0.05) from that of the 223 heifers that ovulated on time (44.8%). In a second experiment, no differences were detected between 15 heifers with delayed ovulation and 15 animals that ovulated on time with respect to their progesterone concentrations during the first 8 days post insemination, indicating that delayed ovulation is not associated with delayed luteinization or subnormal early luteal function. In the third experiment, the conception rate of 126 repeat-breeder heifers that were treated with hCG at the time of insemination was 26.7%; the conception rate of 101 repeat-breeder heifers that were inseminated twice, at 12 and 24 hours after the onset of estrus, was 34.6%; and the conception rate of 105 repeat-breeder heifers which were not treated with hCG and which were inseminated only once was 30.5% (P>0.05) It is concluded that delayed ovulation is not an important cause of infertility and does not constitute an important component of the repeat-breeding syndrome in Holstein heifers.

PREDICTIVE VALUE OF PALPATION PER RECTUM FOR DETECTION OF THE CL IN ZEBU CATTLE AS EVALUATED BY PROGESTERONE CONCENTRATIONS AND ULTRASONOGRAPHY

The main objective of this study was to calculate the predictive value of the positive and negative diagnosis of the presence of a corpus luteum by palpation per rectum in Zebu cattle. Gyr breed (n = 16) heifers were palpated by a veterinarian every 3 d during March and April, and every 5 days during May. The presence or absence of a CL was recorded for 442 examinations. A blood sample was obtained after each examination, and a functional CL was considered to be present if plasma progesterone concentrations were above 1 ng/ml. Progesterone results were used as the reference to calculate the sensitivity and specificity of palpation per rectum for the diagnosis of a functional CL on different months. Predictive values for the positive and negative diagnoses were calculated using different hypothetical rates of the prevalence of CL. To evaluate the morphological basis of an erroneous diagnosis, ultrasonographic scanning of the ovaries was performed in 264 cases and the physical presence or absence of a CL was registered. The calculations were repeated using the ultrasonographic findings as the reference to evaluate the sensitivity and specificity of the diagnosis of a structural CL by per rectum palpation. Several heifers were not cyclic in March but began to cycle during the study. Thus, the prevalence of both functional and structural CL increased with time. As a result, the proportion of palpation diagnoses classified as true positives increased in May, while the proportion of true negatives decreased. Although the values of sensitivity and specificity remained constant from month to month, the predictive values changed markedly in May, when the predictive value of the positive test increased while the predictive value of the negative test decreased. These changes were due to differences in the prevalence of a CL and not to differences in the palpable characteristics of CLs in different months. The prevalence of a structural CL was always higher than that of a functional CL. The Cohens Kappa test for concordance revealed better correspondence between palpation per rectum and ultrasound (k = 0.82) than between progesterone and either ultrasound (k = 0.68) or palpation (k = 0.66). These results show that a large proportion of the errors imputed to deficient palpation when progesterone concentrations are used as a reference are in fact the result of lack of correspondence between the physical and functional presence of a CL.

Luteogenic and luteotropic effects of eCG during pregnancy in the mare.

The role of eCG during pregnancy was evaluated through the study of the temporal relationships between changes in eCG and progesterone concentrations and the formation of supplementary corpora lutea (SCL) in mares impregnated with donkey semen (mule pregnancies) or with horse semen (equine pregnancies). Concentrations of eCG were higher (p<0.01) in equine than in mule pregnancies between weeks 6.5 and 13. Progesterone concentrations were higher in equine than in mule pregnancies between weeks 9 and 17. All animals developed at least one SCL, but more SCL accumulated during equine pregnancies than during mule pregnancies (1.9 ± 0.2 vs 1.2 ± 0.1; p<0.01). In equine pregnancies, the mares that formed a second SCL had higher eCG concentrations (p<0.05) during the two weeks preceding its formation than those mares remaining with only one SCL. Mares that formed a third SCL had higher (p<0.5) eCG levels than those remaining with one or two SCL. Mares with equine pregnancies that formed three SCL had higher progesterone concentrations (p<0.05) than those that formed only one or two SCL. No differences were found in progesterone or eCG concentrations between mares with mule pregnancies that accumulated different numbers of SCL during pregnancy (p>0.05). It is concluded that eCG stimulates both the development of new SCL and the function of existing CL. While these effects are clearly expressed in mares impregnated by horses, the low eCG concentrations during mule pregnancies reduce the impact of this hormone on CL formation and function.

Endocrine alterations around the time of abortion in mares impregnated with donkey or horse semen

The objective of this study was to monitor and compare the concentrations of equine chorionic gonadotropin (eCG), progesterone and estrone sulphate during normal and failed pregnancies of mares impregnated with donkey or horse semen, relating their individual endocrine profiles to the time of pregnancy loss, and to the histopathologic findings in the aborted fetuses and placenta. Mares (n=54) were used, 32 of them impregnated with donkey semen and 22 impregnated with horse semen. Blood samples were taken twice a week from Day 35 to 120 of pregnancy. Ultrasonographic observations of the fetus were carried out twice a week. The incidence of abortion in mares impregnated with donkey semen (30%) was greater (P<0.05) than the 5% observed in mares impregnated with horse semen. From Week 8 to the end of the sampling period, the mean progesterone concentrations of mares with normal mule pregnancies were less (P<0.05) than those of mares with normal pregnancies with equine fetuses. The concentrations of eCG were less (P<0.05) in mule pregnancies from Week 6. Estrone sulphate concentrations were only different (P<0.05) between types of pregnancy on Weeks 13 and 14, being in this case greater with the mule pregnancies. Most of the abortions of mule fetuses were associated with lesser progesterone concentrations than the average for mares with successful mule pregnancies. Four of the abortions of mule fetuses and the only abortion of horse fetus occurred in mares with lesser progesterone and very low eCG concentrations, and were classified as caused by luteal impairment secondary to eCG deficiency; estrone sulphate concentrations were less than normal or absent before these abortions. Two mares aborted after several weeks of low progesterone concentrations in the presence of eCG concentrations that were normal for mule pregnancies, suggesting primary luteal deficiency. In three mares carrying a mule fetus, the concentrations of progesterone and estrone sulphate decreased abruptly immediately before fetal death, suggesting luteolysis due to active prostaglandin F2 alpha (PGF2alpha) secretion. It is concluded that the greater incidence of abortion in mares impregnated by donkeys is associated with different kinds of luteal malfunction. Deficiency of eCG may be a primary cause of many of these cases, either by failing to stimulate enough luteal progesterone secretion and/or by failing to protect the corpora lutea (CL) of pregnancy from endogenous PGF2alpha secretion.

Evaluation of the incorporation of GnRH into a superovulatory regimen for Zebu cattle

The objective of this study was to evaluate the utilization of gonadotropin releasing hormone (GnRH) as part of a superovulatory regimen for Zebu cattle. Forty Zebu cows were superovulated with 40 mg of follicle stimulating hormone-pituitary (FSH-P) divided in eight fractions of 5 mg injected at 12-h intervals. Luteolysis was induced with 15 mg of luprostiol injected at 48 h after the first injection of FSH-P. Half of the animals were injected with 200 ug of GnRH 3 h after the onset of standing estrus. The other 20 animals were not injected with GnRH. All the cows were inseminated three times at 12-h intervals, starting at the time of standing estrus. Embryos were recovered nonsurgically 7 d after the last insemination. Palpation per rectum performed immediately after collection of the embryos did not show differences in the number of corpora lutea between groups (P > 0.05). Likewise, there were no significant differences between treatments with respect to the total number of embryos plus ova, total number of embryos, or the number of transferable embryos recovered (P>0.05). The number of blastocysts, morulae, degenerated morulae and unfertilized ova was similar for the two groups. It is concluded that the incorporation of GnRH into a part of the superovulatory treatment for Zebu cattle does not improve the results of such treatment.

Effect of time and temperature of incubation of heparinized caprine blood on concentrations of progesterone detected in plasma

Blood samples from 12 pregnant does were collected in flasks containing heparin. Each sample was divided into 31 aliquots, the first of which was immediately centrifuged for plasma separation. Fifteen of the remaining aliquots from each goat were kept at 17 degrees C, while the remaining is were refrigerated at 4 degrees C. Centrifugation of these aliquots was carried out at half-hour intervals until completion of 6 h, and then at 8, 12 and 24 h post collection. The mean concentration of progesterone in the aliquots centrifuged initially was 4.0 +/- 0.3 ng/ml, and it was not significantly altered during incubation of the blood at 17 degrees C. In contrast, the concentrations of progesterone increased significantly to 5.0 +/- 0.3 (P < 0.05) during the first hour of incubation at 4 degrees C, remaining elevated most of the time. The stability of progesterone during incubation of heparinized caprine blood at 17 degrees C indicates that immediate centrifugation is not necessary. Refrigeration during the storage of blood samples is not recommended since progesterone levels are altered, probably due to separation of progesterone from the membranes of red blood cells during the hemolysis that is caused by incubation at low temperatures.