Assessment of progesterone concentration using enzymeimmunoassay, for early pregnancy diagnosis in sheep and goats.

The objective of this study was to determine a value of serum progesterone (P4) concentration, assessed using an enzymeimmunoassay (EIA), for the early distinction between pregnant and non-pregnant ewes and goats. Adult, non-lactating ewes of Chios (n=53), Berrichon (n=30) and Sfakia (n=45) breeds were synchronized during the breeding season with progestagens and gonadotrophins and mated to fertile rams (Experiment I). Adult, lactating goats of Swiss breeds (Alpine and Saanen, n=104) and indigenous Greek breed (n=45) were synchronized during the transitional season with progestagens, PGF2alpha and gonadotrophins. Cervical artificial insemination (AI) with fresh semen was applied once, 42-44 h after sponge removal (Experiment II). Jugular blood samples were collected on day 19 after sponge removal (ewes) or on day 21 after AI (goats) and serum P4 concentration was determined by EIA. Progesterone concentrations >/=1.0, >/=1.5, >/=2.5 and >/=4.0 ng/ml were tested as indicative of pregnancy. Pregnancy diagnosis was verified on birth. In the case of sheep, using a discriminatory level of 2.5 ng/ml, overall accuracy of pregnancy diagnosis was 91.4% and predictive value of negative and positive diagnoses were 98.3 and 85.3%, respectively. In the case of goats, predictive value of negative diagnosis was 95.8 and 94.0% and predictive value of positive diagnosis 71.3 and 71.7%, for 1.5 and 2.5 ng/ml, respectively; overall accuracy was 79.2% using either level. The other discriminatory levels tested did not improve these results. A significant positive correlation was observed between P4 concentration and the number of lambs or kids born, and further analysis indicated that this relationship is not a simple linear function. Based on the results of this study, P4 concentrations of 2.5 ng/ml in the case of ewes and 1.5-2.5 ng/ml in the case of goats, determined with EIA, are proposed as discriminatory levels between pregnant and non-pregnant animals, at an interval of one oestrous cycle after service.

Progesterone concentration as an indicator of ovarian response to superovulation in Chios ewes.

We investigated the prediction of the ovarian response to superovulation using progesterone (P4) determination in Chios ewes. During the estrus period. estrus synchronization and multiple ovulations were induced in 100 non-pregnant, non-lactating Chios ewes by a combination of FGA-impregnated intravaginal sponges and 8.8 mg of ovine FSH. Laparoscopic insemination was conducted 24-28 h after the onset of estrus. A concentration of P4 was determined on Day 5 of the estrous cycle and on Day 6 the ovarian response was evaluated by counting the corpus lutea (CL); subsequently, embryo collection was performed. According to the response of their ovaries, ewes were allocated into four groups: A (n = 30); B (n = 37); C (n = 22); D (n = 11), with minimal (0-3 CL), moderate (4-8 CL), good (9-13 CL) or extreme (> 13 CL) ovarian response, respectively. In groups C and D, the mean blood serum P4 concentration (23.2 and 27.3 ng/ml, respectively) was higher (P < 0.001) than that in groups A and B (4.6 and 13.1 ng/ml, respectively); no difference was detected in blood P4 concentration between groups C and D. A strong linear relation (F < 0.00005) was found between blood P4 concentration and the number of CL, as well as between blood P4 and a dummy variable corresponding to poor (< 4 CL) or moderate/good/extreme ovarian response (>3 CL). Our results indicate that based on blood P4 measurement, it is feasible to identify ewes that should show the highest embryo recovery, while it is impossible to predict the exact number of CL formed.

Reproductive efficiency of three estrus synchronization schemes comprising fixed-time insemination in dairy cows.

The objective of this field trial was to compare the odds of pregnancy at 25 days (P25) and at 60 days (P60), of embryonic death (ED) between 25 and 60 days, and of non-pregnant cows returning to heat 20-25 days after artificial insemination (AI). Three fixed-time insemination protocols involving combined administration of either double gonadotrophin-releasing hormone (GnRH) and prostaglandins (A) or GnRH, human chorionic gonadotrophin (hCG) and prostaglandins (B), or double treatment with prostaglandins (C) were used. Cows included in the trial were selected from five herds, comprising a total of 735 (herd size range 85-250, median 120) Holstein dairy cows. Animals used for the study were multiparous, 4-7 years of age, had expressed at least one overt postpartum heat, were between 60 and 120 days of lactation and had at least one palpable corpus luteum and no gross morphological anomalies of their reproductive tracts. They were randomly allocated to receive treatment A (n = 89), B (n = 93), or C (n = 65). Pregnancy of cows not observed in heat 18-24 days post AI was diagnosed by transrectal ultrasonography at 25-30 days and confirmed by rectal palpation at 60-75 days post AI. Those that were detected in heat in the normal period post AI were re-inseminated. No differences were detected among the three treatment groups concerning the odds of P25 (P = 0.5), P60 (P = 0.2) and ED (P = 0.13). In contrast, non-pregnant cows that were administered treatment A were less likely to exhibit estrous signs in the normal period post AI than those administered treatments B (P = 0.02) or C (P = 0.005).

Sexual activity and body and testis growth in prepubertal ram lambs of Friesland, Chios, Karagouniki and Serres dairy sheep in Greece.

Sexual activity, body size and testis growth in the prepubertal period (120-210days of age) were studied in five male lambs of each of four dairy sheep breeds. Sexual responses developed earlier in Friesland than in Chios and Karagouniki and latest in Serres ram lambs. Friesland lambs expressed interest in females at 127days of age and 34kg live weight 18-36days younger and 3.4-6.3kg lighter than the three Greek breeds. Puberty (first mounting with ejaculation occurred) of ram lambs appeared to be more closely associated with body weight than with chronological age. Age at puberty averaged 179days in Friesland, 8, 10 and 30days younger (P<0.05) than in Karagouniki, Chios and Serres, respectively. On the other hand, weight at puberty averaged 50kg for Friesland and Chios compared to about 44kg for the two other breeds (P<0.05). In the four breeds studied there was a 41-52days interval between the time when lambs first expressed interest in females and the time puberty occurred, the longest was that of Friesland lambs. There was a significant breed difference in scrotal circumference. At puberty the scrotal circumference was 33.9cm in Friesland lambs, 4-6cm greater than in the three Greek breeds. Friesland ram lambs had a greater scrotal circumference throughout the entire period studied.

Effect of alpha-tocopherol on plasma testosterone and plasminogen activator activity or inhibition in ram spermatozoa.

The objective of this study was to evaluate the effect of alpha-tocopherol on blood testosterone and specific proteolytic enzymes in spermatozoa and seminal plasma, with final aim the increase of sperm fertilizing ability with a nutritional supplement. The effect of alpha-tocopherol supplementation on testosterone parameters (mean value, basal level, peak number, mean concentration value during peaks, peak amplitude, peak duration) and plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) of spermatozoa and seminal plasma was studied in the ram during autumn (estrous period for the sheep in Greece) and spring (anestrous period). Treated animals showed a marked increase in serum alpha-tocopherol. Testosterone parameters were not affected by the alpha-tocopherol in either autumn or spring, however, the spermatozoal PAA and PAI (anti-tPA) were increased in the spring but not in autumn. These enzymes and their inhibitors are normally increased in autumn (the breeding season) and low in spring. If PAA can improve the fertilizing ability of spermatozoa in the spring, our finding may mean that a nutritional supplement, such as alpha-tocopherol, could provide rams for an accelerated onset of the breeding season in the ewe.

Formation of a secondary corpus luteum after ultrasound-guided follicular aspiration in cows.

This paper reports the observed formation of a secondary corpus luteum (CL) in the presence of the cyclic corpus luteum, on the ovaries of a cow after ultrasound-guided follicular aspiration for oocyte recovery. The secondary structure, although smaller and lighter (4.97 g vs. 6.02 g) than the natural one, had the typical macroscopic appearance of a corpus luteum. Histological examination of the structure using electron microscopy revealed typical structural features of a natural CL. Mean tissue progesterone concentration was significantly lower in the secondary CL (31.15 +/- 3.11 compared with 58.29 +/- 6.32 micrograms/g tissue of the cyclic CL) and oestradiol-17 beta significantly higher than in the natural CL (108 +/- 11.6 compared with 74.2 +/- 7.81 pg/g tissue). P450scc and P450(17 alpha) mRNA was detected in both structures while P450arom and full-length mRNA FSH receptor were detected only in the secondary structure.

Ram seminal plasma and fertility: results from an ongoing field study.

The effects of partial replacement of ram semen diluent with ram seminal plasma on the fertility of ewes were studied. Crossbred Chios ewes (n = 152) were assigned to six groups. The oestrous cycles of the ewes were synchronised at the peak (Groups A, B, C and D) and at the end (Groups E and F) of the breeding season by means of intravaginal sponges impregnated with fluorogestone acetate (FGA) for 14 days. Four hundred IU of PMSG were injected intramuscularly at the time of sponge removal. Ewes of Groups A, C and E were artificially inseminated with ram semen diluted with skim milk extender, while those of Groups B, D and F with ram semen diluted with 50% skim milk and 50% ram seminal plasma. The addition of ram seminal plasma induced a significant increase (P < 0.05) in litter size in Groups B and D when compared with that of Groups A and C (1.85 and 1.88 vs. 1.39 and 1.52, respectively). This increase was not significant when insemination was performed at the end of the breeding season (2.0 vs. 1.4). These results indicate that the addition of seminal plasma can influence the fertility of ewes or the fertilising capacity of extended ram semen to some extent.

Increased plasminogen activator activity and plasminogen activator inhibition in spermatozoa and seminal plasma of the ram after serum gonadotrophin (PMSG) administration. Correlation with the increased level of testosterone in the blood.

After a single injection of serum gonadotrophin (PMSG) at the dose of 15 IU/kg, i.m. into rams testosterone in the plasma of blood showed a significant rise between 4th and 7th day post-injection. At the same time (4th-7th day) the plasminogen activator activity (PAA) in seminal plasma was found to be increased, but the plasminogen activator inhibition (PAI) expressed against t-PA (anti-t-PA) showed an increase between 32nd and 46th day. In spermatozoa a marked increase of PAA was revealed between 32nd and 46th day post-injection, while an increase of PAI (anti-t-PA) was exhibited on the 74th day. Plasmin inhibition (PI) in seminal plasma and spermatozoa showed no change compared to controls. A positive correlation has been found between increased concentrations of testosterone and PAA or PAI (anti-t-PA) in spermatozoa and seminal plasma. The induced increase of PAA in spermatozoa under the effect of testosterone might be of physiological importance, since PAA is localized to sperm membranes and might participate in the whole process of fertilization.