Effect of eCG dose and ovulation induction treatments on embryo recovery and in vitro development post-vitrification in two selected lines of rabbit does

The aim of this work was to evaluate the effect of different doses of eCG administered subcutaneously (0, 50 and 200 IU) and the hormonal induction of ovulation (GnRH or hCG) on embryo recovery and in vitro development of embryos post-vitrification in two selected lines of rabbit does. The two selected lines were line V (selected for the litter size at weaning) and line R (selected for growth rate). Administration of 200 IU of eCG significantly increased ovulation rate (19.2 +/- 1.2 versus 15.5 +/- 1.1 and 12.2 +/- 1.3, and the number of haemorrhagic follicles (13.8+/-1.6 versus 3.8+ /- 1.4 and 3.8 +/- 1.7), but significantly decreased recovery rate (28.8 +/- 6.3 versus 47.7 +/- 5.7 and 48.7 +/- 6.7, 200 IU versus 50 IU and 0 IU eCG, respectively), the number of normal embryos recovered per doe with at least one embryo (5.8 +/- 0.9 versus 8.2 +/- 0.9, 200 IU versus 50 IU eCG doses) and the in vitro development of embryos post-vitrification (51.9% versus 66.1%, 200 IU versus 50 IU eCG doses, respectively). Inducing ovulation with hCG significantly increased ovulation rate when compared with GnRH (17.3 +/- 0.8 versus 13.8+/-1.4), but no significant differences in embryo recovery and embryo development post-vitrification were observed between the two treatments. No significant differences were observed between the two selected lines in ovulation and recovery rates, the number of haemorrhagic follicles and the number of recovered embryos per doe. However, the post-vitrification in vitro rate of development was 59.7% for line R and 51.9% for line V (p < 0.05). It was concluded that the use of 50 IU of eCG subcutaneous with hCG or GnRH prior to embryo cryopreservation programmes in rabbits achieves the best results for embryo recovery, with the best development of recovered embryos post-vitrification.     

Methionine-enkephalin in a porcine endometrial cell line and its responsiveness to potassium depolarization.

Immunoreactive methionine-enkephalin (ir-MENK) has been identified in the porcine uterine fluid and endometrium. Previously, we have established a porcine endometrial cell line of epithelial origin (PE-1) by transfecting primary endometrial cells with temperature sensitive SV40 DNA. The current study was conducted to identify and characterize ir-MENK present in PE-1 cells, and to investigate the effect of KCl depolarization on the kinetics of ir-MENK secretion. PE-1 cells were cultured at 33C until confluency was reached (33C cells), after which they were incubated at 40C for 2 days (40C cells). Ir-MENK in PE-1 cells was analyzed by Sephadex G-15 gel filtration and reverse phase (RP)-HPLC. Analysis of 40C cell extract by Sephadex G-15 and RP-HPLC indicated that the major portion of ir-MENK present in PE-1 cells was eluted at a position similar to that of synthetic MENK. The effect of temperature on ir-MENK synthesis in PE-1 cells was examined by measuring ir-MENK content in 33C and 40C cells over a 14-day culture period. Compared to 33C cells, 40C cells maintained higher and steadier levels of ir-MENK, suggesting that synthesis of ir-MENK is temperature sensitive. KCl stimulated ir-MENK secretion at all concentrations tested (5-60 mM for 60 min), with 30 mM being the optimal concentration. Temporal analysis of ir-MENK secretion showed that incubation for 60 min with 30 mM KCl allowed maximal secretion. Secretion of ir-MENK from PE-1 cells resulted in depletion of ir-MENK in cell content. These results demonstrate that PE-1 cells contain ir-MENK which is biochemically similar to synthetic MENK, PE-1 cells synthesize ir-MENK in a temperature sensitive manner, and these cells secrete ir-MENK upon KCl stimulation.     

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.     

A tissue inhibitor of metalloproteinases and alpha-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown.

Immature female Sprague-Dawley rats were primed with 20 IU eCG at 28 days of age and treated with 10 IU hCG 48 h later. Ovulation followed at 12-14 h. Ovaries were extracted at various times after hCG by use of Triton X-100 and 10 mM CaCl2 (Triton extract) followed by heating to 60 degrees C for 6 min with 0.1 M CaCl2 in 50 mM Tris/0.15 M NaCl, pH 7.5 (heat extract). These extracts were tested for their ability to inhibit tissue metalloproteinases by use of the small uterine metalloproteinase (UMP) of the rat. The ovary contains three plasma-derived inhibitors (alpha 1-macroglobulin [alpha 1 M], alpha 2-macroglobulin [alpha 2 M], and alpha 1 inhibitor3 [alpha 1I3]) and one tissue-derived inhibitor of the tissue inhibitor of metalloproteinases (TIMP) family. alpha 1 I3 was shown to inhibit UMP and rat collagenase. The concentration of the tissue inhibitor rose 5-fold from 0.6 micrograms UMP blocked per gram wet tissue in ovaries not primed with eCG to 3.2 micrograms UMP blocked at 8 h after hCG. Over this same time interval, the sum of alpha 1M + alpha 2M per gram of ovary rose 7-fold from 3.2 to 22.4 micrograms UMP inhibited and alpha 1I3 rose 2-fold from 4.4 to 10.7 micrograms UMP inhibited. The increases in the tissue inhibitor are interpreted to be due to increased synthesis by the tissue, whereas the changes in alpha-macroglobulins are postulated to be due to increased vascularity and increased permeability of the vessels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-culture of mouse embryos with oviduct and uterine cells prepared from mice at different days of pseudopregnancy

Oviduct and uterine cell cultures were prepared from mice at different days of pseudopregnancy and their effects on the development of 1- and 8-cell mouse embryos in co-culture were examined. One-cell mouse embryos in co-culture with oviduct cells from 20 h to 120 h after hCG had a mean (+/- s.e.) cell number of 70.1 +/- 3.6, significantly (P less than 0.001) higher compared with those cultured in Whittingham's T6 medium supplemented with 5% fetal calf serum (T6 + 5% FCS) (30.4 +/- 1.6). Transfer of embryos, at 96 h after hCG, to synchronous pseudopregnant recipients showed that more embryos in oviduct co-culture formed fetuses than those cultured in T6 + 5% FCS. Co-culture of 1-cell embryos with uterine cells did not confer an advantage in cell numbers over T6 + 5% FCS. However, more 8-cell embryos formed blastocyst outgrowths after 100 h in co-culture with uterine cells prepared from mice at Day 3 of pseudopregnancy than with uterine cultures prepared from mice at Day 1 of pseudopregnancy or oviduct cells. In addition, there was further improvement when the Day 3 uterine co-cultures were supplemented with 1 or 10 ng progesterone/ml. These results highlight the importance of the oviduct and uterine cells during the different stages of preimplantation embryo development.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Pregnancies after in-vitro fertilization of cow follicular oocytes, their incubation in rabbit oviduct and their transfer to the cow uterus

Cow embryos fertilized in vitro (1-8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 +/- 4 h after 75 i.u. hCG). Rabbits were killed 99 +/- 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2-8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2-8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.     

Differences in the characteristics and distribution of rat luteal receptors for native and deglycosylated human choriogonadotropin.

Previous work has suggested that rat luteal cells have two populations of LH/hCG receptors that are located in different parts of the cell membrane. The possibility that these two receptor pools may have functional differences has been investigated through examination of the binding and action of native and deglycosylated hCG to different membrane fractions. Ovaries from eCG/hCG-primed immature female rats were separated into 1,000 x g (heavy) and 20,000 x g (light) particulate fractions. Increasing concentrations of NaCl had a biphasic effect on the binding of native and deglycosylated hCG to both membrane fractions, causing an increase in binding at low concentrations and a decrease in binding at higher concentrations. The binding of deglycosylated hCG to both membrane preparations and the binding of native hCG to light-membrane preparations was maximal at approximately the same NaCl concentration (50-65 mM). This was higher than the concentration of NaCl necessary for maximal binding of native hCG to the heavy-membrane preparation. In addition, maximal native hCG binding to this preparation occurred over a broader NaCl concentration range (15-65 mM). Equilibrium binding experiments showed differences in hCG binding to both fractions. In light membranes there were significantly more receptor sites for deglycosylated hCG (11.2 +/- 4.8 fmol/mg ovary) than for native hCG (4.8 +/- 0.7 fmol/mg ovary), with no significant different in affinity. In contrast, in heavy membranes the affinity for deglycosylated hCG (6.30 +/- 0.19.10(9) M-1), was significantly higher than that for native hCG (2.60 +/- 0.13.10(9) M-1), with no significant differences in receptor number.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Immunolocalization of aquaporin 1, 5, and 9 in the female pig reproductive system

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been documented in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs are unknown in the female reproductive system of pigs. In this study, AQP1 immunoreactivity was detected in the capillary endothelium of the ovary. Distinct immunolabeling of capillary endothelium was also observed in the oviduct and uterus. AQP5 was expressed in flattened follicle cells of primordial follicles, granulosa cells of developing ovarian follicles, and muscle cells of the oviduct and uterus. Staining of AQP5 was also observed in the epithelial cells of the oviduct and uterine epithelium. AQP9 immunoreactivity was observed in granulosa cells of developing follicles. AQP9 was also localized in the luminal epithelial cells of the oviduct and uterine epithelia cells. This is, to our knowledge, the first study that shows tissue expression and cellular and subcellular localization of AQPs in the reproductive system of the female pig. Moreover, these results suggest that several subtypes of the AQPs (AQP1, 5, and 9) are involved in regulation of water homeostasis in the reproductive system of gilts.     

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.     

Gelatinase and proteoglycanase activity during the periovulatory period in the rat.

Gelatinase and proteoglycanase are metalloproteinases that govern extracellular matrix remodeling. In the present study, immature rats were primed with eCG (20 IU) and hCG (10 IU). Ovarian gelatinase and proteoglycanase activity were determined at the time of hCG administration (0 h) as well as 4, 8, and 12 h later. Gelatinase and proteoglycanase were extracted by homogenization in Triton and by heating (i.e., heat extraction). An aliquot of the heat extract was reduced and alkylated to destroy metalloproteinase inhibitors. Heat extracts not reduced and alkylated showed low levels of gelatinase and proteoglycanase activity that did not change at the different time points. However, with reduction and alkylation, gelatinolysis increased approximately 4-fold (p less than 0.05) at 4 h, 8 h, and 12 h after hCG priming. Proteoglycanase activity increased approximately 2-fold (p less than 0.05) between 0 and 8 h and declined at 12 h after hCG. The ovarian gelatinolytic activity was due to a metalloproteinase as demonstrated by the inhibition of enzyme activity by phenanthroline and EDTA (97.1 +/- 0.7% and 97.4 +/- 0.6% inhibition respectively). Proteoglycanase activity was not inhibited by phenanthroline (11.5 +/- 3.5%), suggesting that the enzyme activity was not specifically a metal-dependent enzyme. Gelatin gel zymography of the ovarian extracts demonstrated four predominant and distinct gelatin-degrading enzymes of 78, 72, 66, and 62 kDa, similar to the size of gelatinase. The present findings demonstrate a periovulatory increase in ovarian gelatinolytic and proteglycanase activity that may play a pivotal role in connective tissue remodeling associated with ovulation.     

Hastened transport of equine embryos through the oviduct of the mare

The purposes of this experiment were 1) to test the hypothesis that placing rabbit embryos into the mare's uterus would hasten oviduct transport and 2) to determine if placing fluid into the uterus of bred mares on Day 4 and/or Day 5 would subsequently disrupt the mare's pregnancy. The hypothesis that placing rabbit embryos into the mare's uterus would hasten oviduct transport was not supported, since the uterine recovery rate of equine embryos on Day 5 was not significantly higher (P>0.05) for mares receiving rabbit embryos on Day 4 than for mares receiving no uterine infusion on Day 4 (1 10 vs 0 10 , respectively). However, placing fluid into the mare's uterus on Day 4 was apparently responsible for hastened oviduct transport, since mares with media infused into the uterus on Day 4 had a significantly higher (P<0.05) recovery rate of equine embryos on Day 5 than did mares receiving either rabbit embryos or no uterine infusion on Day 4 post ovulation (5 10 vs 1 10 or 0 10 , respectively). The Day-14 pregnancy rate was significantly higher (P<0.05) for mares receiving no uterine infusion on Day 4 or Day 5 than for mares receiving uterine infusion on Day 5 or uterine infusion on both Days 4 and 5 (9 10 vs 4 10 , 2 10 and 0 10 , respectively).     

Expression and localization of luteinizing hormone receptor in the female mouse reproductive tract

The presence of the LH receptor (LHR) in nongonadal tissues of the reproductive tract has been reported, but localization studies have not been performed. Our objectives were to demonstrate the presence of LHR in the reproductive tract and to localize receptor expression. Reproductive age rats and mice were obtained and (125)I-hCG binding assays were performed on membrane preparations from the uterus, ovary, liver, and testis. In situ hybridizations were performed using (35)S-labeled antisense and sense RNA probes prepared from nucleotides 1-591 of the mouse LHR cDNA. Specific hCG binding was detected in membrane preparations from the ovary, uterus, and testis but not in the liver in both the rat and mouse. In the ovary, LHR mRNA was localized in theca cells, large follicles, and corpora lutea as expected. In the uterus, LHR mRNA was expressed in stromal cells of the endometrium and in the uterine serosa. Uterine smooth muscle cells had low levels of expression, and the endometrial epithelium was negative. In the oviduct, high levels of LHR expression were noted on the serosa and in subepithelial cells. Oviductal smooth muscle had low expression, and the epithelium was negative. We conclude that functional, nongonadal LHR are expressed in the mouse reproductive tract. The presence and localization of LHR expression in the mouse reproductive tract lay the foundation for transgenic models to address the physiologic role of these receptors.     

Control of ovulation with a GnRH analog in gilts and sows

Methods for the control of ovulation with GnRH or the GnRH analog D-Phe6 -LHRH (GnRH-A), were evaluated in gilts and sows as the last step in development of a fixed-time Al protocol. This involved 3 field trials using 2,744 gilts (10 units) and 71,628 sows (33 units). In Trial 1, the GnRH-A (75 microg) was given subsequent to treatment with altrenogest for cycle control or eCG for the stimulation of uniform follicle development in gilts. The release of LH was followed by ovulations which commenced within 36.4 +/- 3.3 hr and were terminated at 39.0 +/- 2.8 hr after administration of GnRH-A. This degree of synchronization of ovulations enabled the use of fixed-time AI. Consequently, subsequent to pretreatment with altrenogest and eCG, in 10 production units 1,285 gilts received 50 microg GnRH-A and 1,459 gilts 500 IU hCG serving as positive controls (Trial 2); all the gilts were inseminated 24 and 42 hr after treatment. Pregnancy rate and piglet index (n of piglets per 100 first inseminations) following GnRH-A vs hCG were 78.8% and 779 vs 74.4% and 728, respectively (P < 0.05). In field trials with first litter gilts and multiparous sows (33 units holding from 250 to 6,000 sows), 1,000 IU eCG was used for estrus control after weaning and 25 microg or 50 microg GnRH-A were given 55 to 58 hours after eCG (n = 19,954 and 20,701) (Trial 3). Sows treated during the same time period with 300 microg GnRH plus 300 IU. hCG (n = 30,973) served as positive controls; all sows were inseminated 24 and 42 hours after treatment. Pregnancy rates for 50 microg GnRH-A, 25 microg GnRH-A and 300 microg GnRH plus 300 IU hCG were 83.0%, 81.7% and 80.7%, and the piglet indices 913, 899 and 880, respectively (P < 0.05). Unit size and parity had significant effects on fertility and productivity. In all studies, results with 50 microg GnRH-A were superior. In year-long studies, highest levels of fertility in response to these treatments were seen from December to May.     

Administration of moderate and high doses of gonadotropins to female rats increases ovarian vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression that is associated to vascular hyperpermeability

Convincing evidence supports the role of ovarian-origin vascular endothelial growth factor (VEGF) in inducing vascular permeability (VP) and ascites associated with ovarian hyperstimulation syndrome (OHSS) in mammals, including humans. A circulatory dysfunction has been described in every woman treated with gonadotropins for in vitro fertilization. It is not known, however, whether the action of gonadotropins also includes up-regulation of the VEGF receptor-2 (VEGFR-2) and whether increased VP is also found when milder stimulation is used. Thus, we applied an OHSS animal model to answer these questions. Immature female rats were stimulated with saline (control group) or with high (10 IU of eCG x 4 days + 30 IU hCG, OHSS group) or mild (10 IU of eCG + 10 IU of hCG, mild-stimulation group) doses of gonadotropins. The VP and the expression of whole-VEGF and VEGFR-2 mRNAs were analyzed through time-course experiments (0, 24, 48, and 96 h after hCG). Although eCG increased VP and the expression of VEGF and VEGFR-2 mRNAs in the ovaries of both mild- and OHSS-stimulated animals, hCG further augmented these parameters and produced the highest values after 48 h. A linear correlation was found between increased expression of VEGF and VEGFR-2 mRNAs and enhanced VP in both mild and OHSS groups. Immunohistochemistry showed the presence of VEGF and VEGFR-2 in the granulosa-lutein and endothelial cells of the entire corpus luteum. These studies confirm that in hyperstimulated animals as well as in mildly treated rats, VEGF and VEGFR-2 are overexpressed and associated with an increase in VP, which may be responsible for the accumulation of ascitic fluid in the syndrome.     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

Immunocytochemical localization of catechol-O-methyltransferase in the oviduct and in macrophages in corpora lutea of rat

Summary Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) was localized in rat ovary, oviduct, and uterus using immunocytochemical methods. Immunoreactive deposits were found in the cytoplasm of macrophages in the ovary, epithelial cells of the oviduct, and glandular epithelial cells of the non-pregnant uterus. The pattern of localization observed in the extraneuronal elements suggests that enzyme may function in extraneuronal inactivation of catechols in the ovary, oviduct, and uterus.     

Effect of administration of eCG to postpartum cows on folliculogenesis in the ovary ipsilateral to the previously gravid uterine horn and uterine involution

This study tested the hypothesis that increased oestradiol secretion by large follicles in the ovary ipsilateral to the previously gravid uterine horn has a local effect to increase the rate of uterine involution. Cows were administered an i.m. water placebo (n = 19), 250 iu equine chorionic gonadotrophin (eCG) (n = 18) or 750 iu eCG (n = 20) 14 days post partum (day 0). Transrectal ultrasonography at the time of treatment and 2, 4 and 6 days later monitored uterine horn diameter and ovarian structures. Blood samples collected contemporaneously were assayed for 15-keto-13, 14-dihydro-prostaglandin F(2alpha) and oestradiol concentration. For control cows, accumulated diameter of the largest follicle in the ovary ipsilateral to the previously gravid uterine horn, compared with the contralateral ovary, was smaller on day 0 (P < 0.05) and days 2, 4 and 6 (P < 0.001). There were no significant differences in the eCG-treated animals. There were 12, 8 and 11 cows with a plasma oestradiol concentration < 1 pg ml(-1) on day 0 in the control, 250 and 750 iu eCG treatment groups, respectively. For control cows, the peripheral oestradiol concentrations were higher on day 6 compared with days 0, 2 and 4 (P < 0.05); for cows treated with 250 iu eCG, concentrations were higher on days 4 and 6 compared with day 0 (P < 0.05); and for cows treated with 750 iu eCG, concentrations were higher on days 2 and 4 compared with day 0 (P < 0.01). Treatment with eCG, or the presence of a follicle > 8 mm in diameter in the ovary ipsilateral to the previously gravid uterine horn, did not affect the rate of uterine involution or plasma 15-keto-13,14-dihydro-prostaglandin F(2alpha) concentration. In conclusion, administration of eCG to increase follicular growth and oestradiol production overcame the inhibition of follicular growth in the ovary ipsilateral to the previously gravid uterine horn, but did not affect uterine involution.