The negative effect of repeated equine chorionic gonadotropin treatment on subsequent fertility in Alpine goats is due to a humoral immune response involving the major histocompatibility complex

In dairy goats, the use of eCG as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination. However, repeated eCG treatments are followed by decreased fertility in goats inseminated at a fixed time after treatment. In this report, we show the presence of anti-eCG antibodies in plasma of treated goats. A 500 IU eCG injection induces a humoral response, with variable concentrations of anti-eCG antibody being produced in individual goats. The analysis of successive anti-eCG immune responses over several years has demonstrated the existence of different populations of goats, defined as low, medium, and high responders. By the use of two caprine microsatellites located inside (OLADRB) and outside (BM1258) the major histocompatibility complex (MHC), a significant association (p < 0.05) between the anti-eCG antibody response and some MHC-DRB alleles was found. Goats with high antibody concentrations at the time of eCG injection (> 2.5 microg/ml) exhibited a much lower kidding rate than did other females (41.3% vs. 66.7%). Lower fertility of these goats, inseminated at a fixed time after eCG treatment, might be due to the observed delay in estrus occurrence and the preovulatory LH surge.     

The effect of lecirelin (GnRH) dosage on the reproductive performance of nulliparous and lactating rabbit does

The present study attempted to evaluate the effect of different doses of lecirelin intramuscularly administered at the moment of insemination on parameters such as the conception rate, the total number of rabbits per litter, the number of stillborns per litter and the abortion rate in nulliparous and lactating crossbred rabbit does. The experiment was performed with 295 crossbred female rabbits Hyplus strain PS 19 that were housed in a rabbit farm. Six days before insemination, the light-dark cycles were as follows: 16-h light (70 lx and more):8-h dark, and all does were subcutaneously injected with 25 IU of eCG 48 h before insemination. The doses of lecirelin were as follows: 0.05, 0.1, 0.2, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 microg/doe, and were tested in both nulliparous and lactating female does; the doses were applied at the moment of insemination. The conception rate in nulliparous does ranged significantly from 10.0% (0.05 microg) to 89.5% (1.5 microg). The conception rate in lactating does was the lowest at a dose of 0.05 microg (10.0%), as compared with the doses starting from 0.3 microg, and was confirmed statistically significant. The lowest number of rabbits per litter was found at a lecirelin dose of 0.1 microg (6.64). This finding was statistically significant, as compared with doses equal to or exceeding 0.3 microg. No statistical differences were found for doses ranging from 0.3 to 4.0 microg in nulliparous does (9.06 versus 10.29, respectively). The lowest number of rabbits per litter in lactating does was found at a dose of 0.05 microg (8.28) while the highest number of rabbits was detected at a dose of 4.0 microg (10.73), without statistically significant differences among individual doses. Generally, the number of rabbits per litter in lactating rabbit does was higher than that in nulliparous does (P<0.05). One negative finding is that abortions occurred in lactating does at the intramuscular application of a dose equal to or exceeding 1.5 microg, and in nulliparous does at a dose equal to or exceeding 2.0 microg.     

Four years of induction/synchronization of estrus in dairy goats: effect on the evolution of eCG binding rate in relation with the parameters of reproduction

Ninety-eight Alpine goats of two herds were followed over 4 years in a program of annual artificial insemination after estrus induction/synchronization, including progestagen administration (vaginal sponge) followed by prostaglandin analog and equine chorionic gonadotrophin (eCG) 48 h before sponge removal. Goats were sampled every 4 hours from the 16th to the 56th following sponge removal, for determination of LH surge and tested for estrus by the presence of a buck. Seven days after AI, endoscopic examination of the ovaries was performed to determine the number of corpus lutea. Pregnancy diagnosis was performed at day 21-22 post AI by determination of plasma progesterone and at day 40-45 by ultrasonography. Parturition, number and sex of kids were recorded. All the goats were sampled before and after each treatment, for anti-eCG antibodies screening. Statistical analysis of the results clearly established a significant effect of the treatments on anti-eCG antibodies. Time of estrus and LH surge were significantly different between herd. The antibodies significantly delayed the time of coming out of estrus as well as the time of LH surge. Two antagonistic effects were evidenced: first, the delayed of time of estrus and time of LH surge in relation with the immune reaction to eCG; secondly, the ahead of time of estrus and time of LH surge during the years of treatment, identical to both herd. The antibodies negatively influenced the percentage of ovulating females as well as kidding rate. Finally, no effect of antibodies on prolificacy was found.     

Oestrus synchronisation of rabbit does at early post-partum by doe-litter separation or ECG injection: Reproductive parameters and endocrine profiles

Inseminating rabbit does at early post-partum, in combination with early weaning, can increase prolificacy (total kits born and still born per parturition) and decrease parturition intervals. Oestrus synchronisation increases fertility and prolificacy, while decreasing the number of inseminations required for gestation. However, little is known about the effectiveness of different oestrus synchronisation methods at early post-partum. In this study, does (n = 138) were artificially inseminated nine times (over a period of 1 year, kits weaned at 25 days), on day 4 post-partum after separation from the litter (for 48 or 24 h) or 48 h after 25 UI eCG injection. Plasma levels of prolactin and estradiol were also evaluated in a subsample of 12 multiparous lactating does per treatment, on days 2, 3 and 4 post-partum. The three treatments increased overall fertility of multiparous females compared to controls (which were not synchronised), but there were no differences among treatments in total kits born or stillborn. Does treated with eCG had a higher culling rate. The interval between parturitions and the number of inseminations required for gestation tended to decrease with increasing number of inseminations. In lactating does, there was an interaction between treatment and insemination order. Fertility decreased with increasing inseminations in eCG does but tended to increase above control values in the separated does until the fourth insemination. Control lactating does had significantly less kits per parturition compared to treatments, but eCG lactating does had more stillborn kits. Oestradiol levels increased on day 4 post-partum in all synchronised lactating does (and immediately before artificial insemination in 48 h doe-litter separation), so ovarian activity could be stimulated at early post-partum using all treatments. However, the increase could not be explained by prolactin levels, since there were no effects of suckling absence on plasma prolactin in separated does. In conclusion, separating does from the litter before insemination can be just as effective as eCG treatment, especially during for the first four inseminations.     

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.     

Humoral immune response to equine chorionic gonadotropin in ewes: association with major histocompatibility complex and interference with subsequent fertility.

In dairy ewes, the use of eCG as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination (AI). In this report we show the presence of anti-eCG antibodies in plasma of treated ewes. The major histocompatibility complex (MHC) was involved in the individual variability of the humoral immune responses to eCG. We found significant associations between the anti-eCG response phenotype and some MHC class II alleles. The low immune response phenotype was associated with one MHC class II allele only in Lacaune ewes, and the high immune response phenotype was associated with one MHC class II allele both in Manech and in Lacaune ewes. In herds, the impact of residual anti-eCG antibodies on subsequent fertility after AI seems minimal because of an indirect elimination of high-responder ewes from AI breeding. Therefore, the true magnitude of the association between residual anti-eCG antibody concentration and fertility has been underestimated. An additional experiment without any high-responder female elimination showed a significant correlation between high residual antibody concentrations and lower lambing rate after AI at a fixed time, possibly because of a delayed preovulatory LH surge. The results suggest that anti-eCG antibody concentration is one risk factor for infertility after AI.     

Influence of hormonal and nonhormonal estrus synchronization methods on follicular and oocyte quality in primiparous lactating does at early postpartum period

High-yield lactating does need effective estrus synchronization methods to improve their reproductive outcome by enhancing ovarian function. The aim of the current work was to analyze ovarian follicular and oocyte characteristics of hormonal and nonhormonal estrus synchronization regimes in primiparous lactating rabbit does (Oryctolagus cuniculus) in the early postpartum period (Day 11). Females were randomly treated with either (1) a hormonal standard treatment with 25 IU equine chorionic gonadotropin (eCG) 48 h before artificial insemination (eCG group) or (2) an alternative nonhormonal treatment consisting of doe-litter separation 24 h before artificial insemination (Bio group). No significant differences were found in serum estradiol and progesterone concentrations between experimental groups. During the histologic study, the Bio group presented a higher number of primordial (P < 0.05) and primary follicles (P = 0.07) compared with that of the eCG group, whereas secondary and antral follicular populations were similar. Rates of late atretic follicles assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique were not different between treatments, but the eCG group showed a significantly higher number of mid-atretic follicles compared with that of the Bio group. Nuclear in vitro oocyte maturation (IVM), measured as metaphase II rate, and in vitro steroidogenic response of cumulus-oocyte complexes, measured by ELISA, did not show significant differences between treatments. However, confocal study showed that cytoplasmic maturation of oocytes, in terms of cortical granule migration rate, was significantly higher in the Bio group compared with that after the eCG treatment. In conclusion, transient doe-litter separation seems to improve ovarian response in terms of follicular health and oocyte competence compared with that after the eCG treatment. Therefore, a 24-h-long transient weaning could be an alternative nonhormonal method for synchronizing estrus in primiparous lactating rabbit does inseminated in the early postpartum period.     

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep

This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n = 19) or simplified (n = 19) superovulatory treatment up to three times at intervals of ≥ 50 d. The onset of estrus was 5 h earlier (P < 0.05) among ewes that received the eCG/FSH protocol (25.2 ± 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ± 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P < 0.05 to P < 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ± 2.3 and 13.7 ± 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P < 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies.     

Preliminary report on induction of estrus with multiple eCG injections in Colored Mohair goats during the anestrus season

This trial was conducted to evaluate the effectiveness of multiple eCG injections in the induction of estrus and pregnancy in Colored Mohair goats during the anestrus season. It was also aimed to determine total dose of eCG required for induction of estrus. Ten multiparous and lactating goats were used. The goats were randomly divided into two groups and treatments were started on May 22. Group eCG (n=5) was treated with eCG intramuscularly for 6 days. Daily dosages of eCG from May 22 to May 27 were 300 IU, 200 IU, 200 IU, 100 IU, 100 IU and 50 IU, respectively. Goats in control group received no treatment. Blood samples were taken from animals in each of the two groups just before and after the beginning of the treatments and serum progesterone concentrations were assayed by RIA. Starting on the fourth day after the first treatments, goats were exposed to fertile bucks twice daily for 30 min to detect standing heat. The estrus goats were allowed to be mated by the bucks. Pregnancies were determined 40 days after mating by real-time ultrasonography. One goat on day 5 and three goats on day 7 exhibited behavioral estrus in eCG group (80%) after the first eCG injection. Three of them (75%) became pregnant. None of the goats in the control group exhibited behavioral estrus. Mean serum progesterone concentrations had prominent elevations indicating ovulation in eCG group, but not in control group, after 20 days from the first treatments. Progesterone concentrations of eCG group were significantly different than those of control group on days 20 and 28 (P<0.05). The results suggest that divided multiple injections of a total 950 IU eCG are effective without progestagen pretreatment in the induction of estrus and obtaining successful pregnancy and live kids in Colored Mohair goats during the anestrus season.     

Effect of different number of frozen spermatozoa inseminated on the reproductive performance of rabbit does

The relationship between the number of frozen spermatozoa inseminated and fertility rate and litter size at birth in rabbit does was investigated. Six hundred artificial inseminations (AI) were performed on multiparous lactating does with three spermatozoa concentrations: 10, 25, 50x10(6)spermatozoa/AI. All the does were synchronized with 20 UI of eCG 2 days before AI. The estimated sexual receptivity was 87%. The freezing-thawing procedure strongly reduced kinetic and functional traits (acrosome integrity, capacitation) of the spermatozoa. The number of spermatozoa inseminated did not affect the reproductive performance: the mean fertility rate and litter size values were 51.5% and 7.6%, respectively. Sexually receptive does (n=522) inseminated with frozen spermatozoa showed a 58.0% fertility rate whereas, non-receptive does (n=78), had a very poor fertility rate (7.8%).     

Synchronization of estrus and ovulation in sows not conceiving in a scheduled fixed-time insemination program

A field study was conducted to investigate the effectiveness of a treatment with altrenogest, eCG and hCG or the GnRH-analogue D-Phe(6)-LHRH to synchronize estrus and ovulation of sows diagnosed as non-pregnant in order to reintegrate them back into a scheduled fixed-time insemination program. Sows (n=531) diagnosed as non-pregnant by ultrasonography on days 21-35 after insemination were subjected to one of three treatments: (1) 16 mg altrenogest/day/animal orally for 15 days to block follicular growth, followed by injection of 1000 IU eCG intramuscularly (i.m.) 24h after withdrawal of altrenogest to stimulate follicular growth and 500 IU hCG i.m. 78-80 h after eCG to induce ovulation; (2) similar to (1) except that 20mg altrenogest and 800 IU eCG were used and (3) similar to (2) except that 50 microg D-Phe(6)-LHRH was used to induce ovulation. Females were artificially inseminated (AI) twice at 24 and 40 h, respectively, after hCG/D-Phe(6)-LHRH. Success of treatments was checked by ultrasonography of the ovaries. Rates of conception and farrowing (CR, FR), and number of total and live born piglets (TB, LB) were recorded and compared to those of synchronized first served sows. Females had differing ovarian structures prior to treatment. Altrenogest effectively blocked follicular growth in >80% of the females irrespective of dosage, but 16 mg increased the development of polycystic ovarian degeneration. Four to 18% of the females still had corpora lutea after altrenogest. Most females ovulated either between both inseminations or thereafter (P<0.05). Females treated with D-Phe(6)-LHRH tended to ovulate earlier than those injected with hCG. The CR and FR were up to 25% lower for sows diagnosed as non-pregnant than for sows after first service (P<0.05). Among sows diagnosed as non-pregnant the CR was higher in females treated with D-Phe(6)-LHRH (P<0.05). No differences were found in regard to numbers of TB and LB. In conclusion, a treatment with 20mg altrenogest per day per animal, followed by 800 IU eCG and 50 microg the GnRH-analogue D-Phe(6)-LHRH is appropriate to synchronize estrus and ovulation of sows diagnosed as non-pregnant. Whether there might be a need to feed altrenogest for a longer interval of 18 days has to be investigated.     

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.     

Effect of sperm numbers and time of insemination relative to ovulation on sow fertility

We examined the effect of inseminating mixed parity sows (n = 231) once with fewer sperm at different times relative to ovulation. Lactation length was 19 days and sows received an IM injection of 600 IU equine chorionic gonadotrophin (eCG) 12 h before weaning. At 80 h after eCG injection, sows received an IM injection of 5 mg porcine luteinizing hormone (pLH). Predicted time of ovulation (PTO) was 38 h after pLH injection. Sows were assigned by parity to receive a single transcervical artificial insemination (AI) at either 6 or 24 h before PTO with semen doses containing either 2.5 or 1.25 × 10⁹ sperm. A positive control group of sows (n = 49) was subject to conventional AI 24 and 6 h before PTO. Detection of estrus was performed in the presence of a boar and only sows exhibiting estrous behavior at the assigned time of AI were included in the study. Farrowing rate for sows receiving 2.5 × 10⁹ sperm at 6 h before PTO was greater than that for sows receiving 1.25 × 10⁹ sperm at 24 h before PTO (85% versus 61%, P < 0.05). All other groups were intermediate. There was no effect of time of AI or sperm numbers on subsequent litter size. These data indicate that single insemination of fewer sperm may compromise sow fertility, even when performed transcervically, if not appropriately timed relative to ovulation.     

The effect of equine chorionic gonadotropin (eCG) on pregnancy rates of white-tailed deer following fixed-timed artificial insemination

Control of the white-tailed doe's reproductive cycle is not well documented. The objective was to determine the effects of giving equine chorionic gonadotropin (eCG) at progesterone device removal on fixed time artificial insemination (FTAI) pregnancy rates in white-tailed does. All does (n = 74) were synchronized with a vaginal progesterone implant (CIDR; 0.3 g progesterone), inserted on Day 0 (without regard to stage of estrous cycle), removed 14 days later, and subjected to FTAI, on average, 60 h post-CIDR removal. Of these, 34 were given 200 IU (im) of eCG at CIDR removal. Overall, FTAI pregnancy rate was 50% across 2 yrs (effect of year, P = 0.35). Administration of eCG at CIDR removal did not affect (P = 0.16) pregnancy rate (eCG = 59%; no eCG = 43%). Pregnancy rates were not affected by vulva score or doe disposition. Does that were ≤ 4 yrs old were more likely (P = 0.01) to become pregnant than does > 4 yrs of age. Does inseminated ≥ 60.5 h after CIDR removal were 22 times more likely (P = 0.002) to become pregnant to FTAI than does inseminated < 60.5 h. When frozen-thawed semen was deposited in the cervix or uterus, does were 17 times more likely (P = 0.005) to become pregnant compared with those receiving intravaginal insemination. Fecundity was not different (P = 0.73) across treatment groups (1.6 ± 0.11; no eCG vs. 1.7 ± 0.10; eCG). Furthermore, fecundity of does pregnant to FTAI was not different (P = 0.72) compared with does pregnant to clean-up bucks (1.7 ± 0.08; AI does vs. 1.7 ± 0.09; clean-up bucks). In summary, white-tailed does were successfully inseminated using a 14 days FTAI protocol, eCG may not be essential for acceptable pregnancy rates, and increased pregnancy rates may result when FTAI is done ≥ 60.5 h after progesterone device removal.     

Increase of plasma eCG binding rate after administration of repeated high dose of eCG to cows

Equine chorionic gonadotrophin (eCG) is still used to promote follicular growth in cattle and, more recently with an increased frequency of administration, in ovum pick-up protocols. The aim of this experiment was to verify the possible effect of high frequency of administration on the immune response to eCG. The profiles of eCG binding rate, in the blood of two groups (A, B) of 4 primiparous cross breed beef cows (3-3.5 years old) submitted weekly for 5 to 10 weeks to repeated high doses (1000-2000 IU) of equine chorionic gonadotrophin, are presented in this paper. A sensitive radiometric method was used to detect antibodies in plasma. The profiles clearly indicated a marked increase of eCG binding rate after 3 to 5 injections of the exogenous hormone to the females. The statistical analysis of the results established that treatments induced a significant increase (P < 0.01) in binding rates after 6 and 3 injections in group A and B respectively. These binding rates remained elevated for at least 1 week following the last injection and decreased afterwards. The values of plasma binding rates following repeated eCG administration differed significantly between groups (0.90+/-1.04 and 1.04+/-0.11 for groups A and B before treatment versus 11.77+/-0.92, 6.70+/-0.85 for groups A and B after treatment, P < 0.01) and from one cow to another (P < 0.01) with some cows presenting no significant immune response while others were more reactive against the hormone (at least 3 injections).     

Ultrasonic study of follicular dynamics following superovulation in German Merino ewes

Ewes are commonly superovulated with a single dose of eCG or multiple doses of pFSH. It would be convenient and less expensive to use a single dose of FSH, but results of various trials have been controversial. We wished to investigate ovarian dynamics using ultrasonography after superovulation with a single dose of pFSH and hMG as compared with a single dose of eCG. Estrus was synchronized during the breeding season with fluorogestone acetate-containing intravaginal sponges in adult German Merino ewes (n = 38). They were superovulated with single doses of pFSH (17 mg; n = 10), hMG (600 IU FSH and 600 IU LH; n = 9) or eCG (1250 IU; n = 10) given at the time of sponge removal, or pFSH (17 mg; n = 9) given 36h before sponge removal. Follicular and luteal development were observed by ultrasonic scanning every 8 h from the gonadotrophin injection until the end of estrus, and then once daily until Day 6 after estrus. Jugular venous blood was collected starting immediately before and 1 h after superovulation treatment, then twice daily until the end of estrus and once daily for the following 7 days. Concentrations of estradiol-17beta (E2) and progesterone (P4) were measured in plasma. Differences in the follicular dynamics of the 4 superovulation groups were obvious. The functional duration of the pFSH action was estimated to last approximately 48 h, whereas eCG and hMG were active for up to 72 h. The diameter of the ovulatory follicles proved to be smaller than it was described for unstimulated ewes. Single applications of pFSH or hMG can induce a superovulatory response, although the post-estrus progesterone profile revealed a high premature luteal regression rate in the different superovulation groups. Premature corpus luteum regression could not be seen by ultrasonography at this early stage of the luteal phase, indicating that the technique may fail to detect these corpora lutea in an embryo transfer program. However, ultrasonography represents a suitable method to observe follicular dynamics following different superovulation regimens in sheep.     

Endocrine and ovarian response after a 2-day controlled suckling and eCG treatment in lactating rabbit does

Synchronization methods are used to obtain higher fertility when artificial insemination (AI) is applied to lactating rabbit does. The most common methods are eCG administration or temporary doe-litter separation. Nevertheless, drawbacks have been reported, such as negative side effects of hormonal treatment in the doe and low litter growth due to absence of suckling, respectively. Recently, improved reproductive performance (without visible consequences on young rabbit growth), has been obtained by applying a 2-day controlled nursing method before AI, by allowing for a 10 min nursing of the litter 24 h of separation. The present study was undertaken to examine the pituitary (PRL, LH, FSH) and the ovarian response (follicle size and number) to those methods. A total of 442 lactating does inseminated on day 11 post-partum were distributed in three experimental groups: 2CN (closing of nest box on day 9, controlled nursing on days 10 and 11), eCG (20 IU administered on day 9 post-partum) and CONTROL (untreated). Blood samples were obtained from 10 does per group at 48, 24 and 0 h before AI, and 1h after AI. Both 2CN and eCG treatments similarly improved sexual receptivity (76.3, 77.5 and 58.2%, respectively; P<0.001) and fertility (63.1, 64.1 and 48.4%, respectively; P<0.05) in lactating does, compared to the CONTROL group. Similar plasma FSH levels in all groups of does and sampling times were observed. Due to the absence of suckling, plasma concentration of PRL on day 10 post-partum in the 2CN group was lower than in the CONTROL group (P<0.05); this endocrine change in PRL levels could explain the better reproductive performances obtained with 2CN treatment. At 1h after exogenous administration of GnRH (at the moment of AI) a high LH response was observed in all groups (P<0.001). Ovaries from 20 rabbits treated in the same way but uninseminated (2CN, n=10; eCG, n=5; CONTROL, n=5 does) were obtained on day 11 post-partum in order to check the morphometric status (weight, width and height) and to make histological and immunohistochemical studies to detect growth hormone receptor (GH-R). As a result, synchronization methods did not show any significant difference in relation to the CONTROL group. However, a small increase in the number of primary follicles was evidenced in the 2CN group with respect to the eCG group, similarly to the CONTROL group (23.0+/-3.7, 9.4+/-4.9 and 14.8+/-4.92 primary follicles, respectively; P=0.1). GH-R immunostaining-presence was more evident in the 2CN and the eCG groups, including primordial follicles and oocytes themselves. Thus, there could have been some direct effects of GH on follicular development, as described in other species. Some ovarian parameters described open new ways to study intra-ovarian mechanism of follicular development in the post-partum period of rabbit does.     

In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.     

Elimination of apoptotic spermatozoa from rabbit insemination dose using annexin V associated with the MACS technique. A preliminary study

The aim of this study was to verify whether the separation and elimination of the apoptotic fraction in rabbit semen using a MACS technique may improve sperm fertility potential and consequently rabbit kindling rate. Semen samples from 25 New Zealand White (NZW) rabbit males were collected using an artificial vagina and evaluated using the CASA system for concentration and motility. For artificial insemination the best 11 bucks were chosen based on motility parameters. Their ejaculates were mixed to make a heterospermic pool and routinely diluted in a commercial insemination diluent (MiniTüb, Tiefenbach, Germany) at a ratio of 1:6. Diluted heterospermic spermatozoa were filtered through a Sartorius filter to wash out seminal plasma, re-diluted in binding buffer (Annexin V Microbead Kit, Miltenyi Biotec, Germany) at a ratio of 1:3.66 and divided into two groups: an experimental group intended for MACS separation and control group without MACS separation. Then hormonally treated females of NZW rabbits were inseminated with fresh doses of filtered heterospermic semen (n = 27; 0.5 ml I.D. per female) and MACS separated semen (n=28; 0.5 ml I.D. per female). Separation and subsequent elimination of apoptotic spermatozoa (positive selection) from the insemination dose (after negative MACS selection) was verified under in vivo conditions on the basis of increased kindling rate in the experimental group in comparison with kindling rate in the control group (81.3% vs. 73.8%). In conclusion, elimination of apoptotic spermatozoa by the use of the MACS technique results in a slight improvement in kindling rate of rabbit does.