Identification of relaxin gene expression and protein localization in the uterine endometrium during early pregnancy in the pig.

The corpus luteum is the primary source of circulating relaxin (RLX) in female pigs. However, a preliminary experiment in our laboratory identified RLX in the uterine endometrium of a day 16 pregnant gilt that had been ovariectomized on day 8 of pregnancy, and the pregnancy maintained via progesterone replacement therapy. Therefore, our objective was to examine the uterus as a potential extraovarian source of RLX during the estrous cycle and early pregnancy in pigs. Reproductive tissues were collected from pregnant (n > or = 3/day) and nonpregnant (n > or = 3/day) gilts on days 10 (n = 6), 12 (n = 6), 14 (n = 6), 16 (n = 8), 18 (n = 6), and 20 (n = 6) of the estrous cycle and pregnancy and on day 42 (n = 2) of gestation. To verify that the RLX identified in uterine tissues was not of ovarian origin, three additional pregnant gilts were ovariectomized on day 8, and the pregnancy was maintained by progesterone replacement therapy until day 16, when the reproductive tissues were collected for immunohistochemistry. The uterine tissues were scored for specific RLX immunostaining and analyzed for the effects day and pregnancy by analysis of variance. Within the uterine endometrium, the luminal epithelium of pregnant pigs contained more RLX than that of nonpregnant pigs (P < or = 0.001). However, there was no difference in either the glandular epithelium or stromal tissues (P > or = 0.10). Increased RLX in the luminal epithelium of pregnant pigs was detected on both days 18 (P < or = 0.05) and 20 (P < or = 0.001) of gestation compared to that in nonpregnant pigs on similar days of the estrous cycle. An effect of day was also observed for RLX immunostaining in the luminal epithelium (P < or = 0.005), but not in glandular or stromal tissues (P > or = 0.10). In both pregnant and nonpregnant pigs, luminal epithelial RLX immunostaining was faint on days 10-12. Thereafter, RLX immunostaining increased on day 14 to reach peak levels on day 16. In nonpregnant pigs, RLX immunostaining dropped to low levels on day 18 and became faint to absent by day 20 of the estrous cycle. In pregnant pigs, RLX immunostaining remained elevated, but appeared to decline slightly by day 20 and became undetectable by day 42 of gestation. RLX immunolocalization in the glandular epithelium was detected on all days in pregnant and nonpregnant pigs, whereas stromal-specific immunostaining was not observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of ovarian steroids on relaxin-induced uterine growth in ovariectomized gilts.

This study examined the influence of ovarian steroids on the uterotropic actions of relaxin (RLX) in ovariectomized prepubertal gilts. Ovariectomized gilts received (im) corn oil (CO), estradiol benzoate (EB), or EB and progesterone (P) for 0-16 days. Steroid administration was patterned to approximate the plasma concentrations of endogenous ovarian steroids observed during 1) the follicular phase (EB), 2) luteal phase (EB+P), and 3) early pregnancy (EB+P+EB). Half of each group also received PBS or 0.5 mg RLX every 6 h for 54 h, coinciding with the final 2 days of the experimental period. After hysterectomy, uterine tissues were analyzed for water, dry matter, protein, DNA, glycosaminoglycans (GAGs), and collagen contents. Administration of EB or P increased uterine weight 5- to 6-fold, but no differences were observed between EB+P- and EB+P+EB-treated gilts. Cotreatment with RLX enhanced steroid-induced uterine growth 40-70%, and RLX stimulated growth in CO- and EB+CO control gilts 2- to 3-fold. The water content of uterine tissues was greater in EB-, EB+P-, and EB+P+EB-treated gilts than in their respective controls, and this response was augmented by RLX in all treatment groups. Administration of steroids stimulated a 4- to 5-fold increase in uterine dry weight compared to that in controls, with responses not differing between EB+P- and EB+P+EB-treated gilts. In all groups, RLX increased uterine dry weight. Protein and DNA contents of uterine tissue increased with steroid treatment, but neither variable differed between EB+P- and EB+P+EB-treated gilts. Administration of RLX, alone or in combination with steroids, increased protein and DNA contents of uterine tissues. The tissue content of GAGs increased in response to steroids, and coadministration of RLX did not alter this response. Although the uterine tissue concentration of collagen was reduced in steroid- and RLX-treated gilts, the collagen content of the uterus was not affected by the various treatments. The results of this study indicate that RLX is a potent stimulator of uterine growth under a variety of steroidal environments. RLX- or steroid-induced uterine growth was manifest by increased water, dry matter, protein, and DNA and GAG contents, but the uterine content of collagen was not affected. The overall growth-promoting effects of EB and the stimulation of DNA accretion by RLX were not observed when gilts were cotreated with P.(ABSTRACT TRUNCATED AT 400 WORDS)

Reproductive biotechnologies: current status in porcine reproduction.

During the past decade, considerable attention has been directed toward the development of reproductive technologies for both research purposes and for more controlled swine reproduction. Artificial insemination is an example of a technology that has continued to be expanded from early use in European countries to the USA and Canada where it is now estimated that a majority of the sows bred are artificially inseminated. In addition, several significant technological advancements have been made in the genetic modification of swine and interest has been generated in the possible use of swine as donors of specific tissues and of organs for the improvement of human health. At the same time, the systems for production of swine for human food continue to undergo major changes including, in some countries, the consolidation of swine into large, integrated units. These swine operations are very receptive to the use of technologies to reduce labor costs as well as a basis for increased production efficiency. Therefore, the combined interest in swine reproductive technologies by both the medical field and the swine industry creates an increased effort for the development of new technologies as well as for the implementation of existing ones. One of the more rapid technological advancements this decade has been the progress in in vitro production (IVP) of swine embryos. Major advancements have been made on the development of procedures for production of large numbers of embryos from oocytes collected at slaughter houses which are then matured (IVM) and fertilized (IVF) in the laboratory. Success in IVP has stimulated increased research in other areas that can be enhanced by the availability of embryos without a requirement for surgical collection from gilts or sows. One example is the combined use of IVF, gender-sorted sperm cells, and embryo transfer to produce offspring of a predicted sex. In a related area, instrumentation for non-surgical embryo transfer has recently been developed that results in significant improvement in this technology. Similar achievements have been gained in cryopreservation of embryos by vitrification. These developments will be reviewed with emphasis on the in vitro production of embryos from immature oocytes.

Development of pig embryos reconstructed by microinjection of cultured fetal fibroblast cells into in vitro matured oocytes.

Nuclear transfer as originally developed for use in amphibians involved microinjecting a nucleus directly into the cytoplasm of the oocyte. A major mammalian modification has been to use cell fusion to introduce the nucleus. Here we report using a microinjection method to introduce small and medium sized fibroblast cells into mature oocytes. Small cells were more likely to result in nuclear formation (30%) than larger cells (15%; P = 0.013). Small, confluent and serum starved cells resulted in nuclear formation more often (P < 0.048) than did cycling cells. The rate of nuclear formation was not dependent upon the media, (NCSU-23 or TL-Hepes without calcium) nor upon the duration of exposure to the media (1 h to 4 h) after microinjection but before activation. While such treatments did not have an effect on nuclear formation, treatment of parthenogenetically activated oocytes with calcium-free TL-Hepes reduced the percentage of blastocysts (P = 0.068. 11.2% vs. 18.3%) and increased the percentage of morula stage embryos (P = 0.007; 27.6% vs. 15.7%) as compared with culture in NCSU. Finally, small confluent cells were used for nuclear transfer and resulted in two presumptive blastocyst stage embryos [2/128 injected or 2/38 (5.3%) successful injections]. These results show that presumptive blastocyst stage embryos can result from microinjection of fibroblast cells to enucleated oocytes and thus may provide a method to create transgenic knockout animals.

Development and expression of the green fluorescent protein in porcine embryos derived from nuclear transfer of transgenic granulosa-derived cells.

Nuclear transfer (NT) techniques have advanced in the last few years, and cloned animals have been produced from somatic cells in several species including pig. In this study we examined the feasibility of using granulosa-derived cells (GCs) as donor cells combined with a microinjection procedure to transfer those nuclei. In vitro matured oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm. Mural GCs infected with an enhanced green fluorescence protein (EGFP) gene were serum-starved (0.5% serum, 7 days), injected directly into cytoplasm of enucleated oocytes and the oocytes were electrically activated. The reconstructed embryos were cultured for 7 days and stained with Hoechst 33342 to determine the number of nuclei. Non-manipulated oocytes were electrically activated and cultured as controls. At 9 h post-activation, the pronuclear formation rates were 78.7+/-3.7% in NT and 97.4+/-4.4% in controls at 9 h post-activation. After 7 days culture, the cleavage rates were 24.5+/-7.2% in NT and 79.3+/-5.6% in controls. The blastocysts formation rates were 4.9+/-2.4% in NT and 26.8+/-3.8% in controls. To examine the effect of activation time on development of NT embryos, oocytes were activated at 0-0.5, 1-2, or 3-4 h post-injection. At 18 h post-activation the pronuclear formation rates were higher (62.5+/-7.3%) in the 3-4 h group as compared to the 0-0.5 h (22.0+/-12.5%) or 1-2h (44.5+/-6.3%) groups (P<0.05). However, the cleavage rates (9.6+/-4.6 to 10.7+/-4.2%) and the blastocysts formation rates (1.2+/-2.4 to 4.9+/-3.7%) were not different among treatments (P>0.05). The mean cell number of blastocysts was 15.7+/-5.7 in NT and 25.3+/-24.7 in controls. Green fluorescence was observed in roughly half of the embryos from the one-cell to the blastocyst stage. These results indicate that granulosa-derived cell nuclei can be remodeled in the cytoplasm of porcine oocytes, and that the reconstructed embryos can develop to the blastocyst stage. In addition, EGFP can be used as a marker for gene expression of donor nuclei.

Induction of parturition in swine with prostaglandin F(2)alpha, estradiol benzoate and oxytocin.

Pregnant sows and gilts were administered either 0, 2.5, 5, 10 or 20 mg prostaglandin F(2)alpha (PGF(2)alpha) intramuscularly on Day 112 or 113 of gestation at 0800 h in an effort to induce parturition. The average interval from PGF(2)alpha injection to farrowing was 55.1 +/- 5.7, 29.4 +/- 3.1, 32.1 +/- 4.6, 27.8 +/- 1.8 and 26.9 +/- 1.1 h for 0, 2.5, 5, 10 and 20 mg, respectively. All PGF(2)alpha treatments increased (P < 0.01) over controls the number of sows farrowing 23 to 33 h after injection. The average gestation length was significantly shorter in treated gilts; however, no detrimental effect on pig performance or pig survivability was observed. A second trial evaluated the effect of a 10-mg dose of PGF(2)alpha on the induction of parturition in sows in order to obtain a majority of sows farrowing within normal working hours (0700 to 1700 h). The interval from injection to farrowing was decreased (P < 0.05) by PGF(2)alpha treatment (66.2 +/- 5.3 vs 28.1 +/- 2.2 h). Fifty-seven percent (P < 0.05) of PGF(2)alpha-treated sows farrowed between 0700 and 1700 h as compared to 13.6% for control sows. A third trial was conducted to examine a sequential treatment of PGF(2)alpha and oxytocin to control the time of parturition more precisely. Sows receiving only 10 mg of PGF(2)alpha farrowed on an average 31.1 +/- 1.4 h after injection. The injection of 40 IU oxytocin 24 to 28 h after PGF(2)alpha decreased (P < 0.05) the interval from PGF(2)alpha to farrowing (28.1 +/- 0.9 h). The addition of oxytocin increased (P < 0.05) the number of sows farrowing within 3 h of injection (33 vs 86% for PGF(2)alpha and PGF(2)alpha + oxytocin treatments, respectively). A fourth trial was designed to determine if the addition of exogenous estradiol benzoate (EB) to a sequential treatment of PGF(2)alpha and oxytocin would improve the predictability and synchronization of the induced parturition. Sows were assigned to receive either saline, 10 mg PGF(2)alpha + 40 IU oxytocin or 10 mg PGF(2)alpha + 5 mg EB + 40 IU oxytocin. The addition of EB reduced (P < 0.01) the variance in the interval from oxytocin to farrowing and added precision to the predicted time of induced parturition.

In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium.

The effect of BSA, caffeine and calcium was studied on the penetration of pig oocytes by frozen-thawed spermatozoa in a modified Tris-buffered medium (mTBM) without added bicarbonate. Pig cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. The COC were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with spermatozoa for 6 h in mTBM containing caffeine (5 mM) and 0.1 or 0.4% BSA (Experiment 1). In Experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0 to 5 mM). In Experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca(2+) (0.5 to 10 mM). Supplementation of mTBM with either 0.1 or 0.4% BSA resulted a high penetration rate with a high polyspermy rate. However, the mean number of spermatozoa per oocyte was significantly higher at 0.4% than at 0.1% BSA. The penetration rate, polyspermy rate and mean number of spermatozoa per oocyte were all significantly higher when 1 to 5 mM caffeine were added to the medium than in caffeine-free medium. No penetration was observed in the presence of 0.5 mM Ca(2+). The penetration rate was significantly increased from 12 to 92% at 2.5 to 10 mM Ca(2+). The mean number of spermatozoa per oocyte did not differ between 2.5 and 5 mM Ca(2+) but increased significantly at 7.5 and 10 mM. These results show the successful in vitro penetration of pig oocytes in a chemically semidefined medium without added bicarbonate. Although BSA and caffeine can modulate the rate of sperm penetration, calcium seems to be an important regulatory ion.

The effect of luteinizing hormone, human chorionic gonadotropin and adrenocorticotropic hormone on puberty in gilts reared in confinement.

Three trials were conducted to determine the effect of human chorionic gonadotropin (HCG), luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) on the incidence of estrus in gilts which were reared in confinement, relocated and exposed to a boar. In trial 1, 33 gilts were given saline or 250 IU HCG at an average age of 191 days and then relocated and observed for estrus twice daily for 10 days. Treatment with HCG did not increase the proportion of gilts that exhibited estrus. In trial 2, 42 gilts were relocated at an average age of 200 days. The gilts were assigned to three treatment groups and injected with saline, 68 microg LH or 1 mg LH. After 10 days of estrous detection, a laparoscopic examination of the ovaries was conducted on all gilts failing to exhibit estrus. In groups 1 to 3, the proportions of gilts exhibiting estrus or ovulating during the 10 days after treatment were 13 of 21, 6 of 10, and 5 of 11, respectively. In trial 3, 12 gilts were relocated to pasture lots, given saline or 80 IU ACTH twice daily for 2 days and checked for estrus for 14 days. The proportions of gilts that exhibited estrus after the administration of saline or ACTH were 4 of 6 and 6 of 6, respectively. The results indicate that the incidence of estrus in gilts reared in confinement, relocated and exposed to a boar was not affected by pre-treatment with exogenous HCG, LH or ACTH.

Estrus and ovulation in gilts fed a synthetic progestogen.

A synthetic progestogen, allyl trenbolone (AT), was fed to sexually mature gilts to determine the effective doses for the control of estrus and ovulation. Gilts were assigned to a control group and 5 treatment groups receiving 10.0, 12.5, 15.0, 17.5 or 20.0 mg of AT mixed in .45 kg of feed/head/day for 18 consecutive days. Ovarian morphology was determined by laparotomy following estrus or at 10 days post-treatment. AT suppressed estrus in all gilts during treatment. Estrus was effectively synchronized in treated gilts. The average interval from withdrawal of progestogen treatment to estrus was 4.5 days for 48 of 50 treated gilts that were in estrus within 10 days after treatment. The average ovulation rate in treated gilts was similar to control gilts. No detrimental side effects, due to treatment, were observed with the possible exception of a slight increase in the incidence of cystic follicles.

Practical considerations for the in vitro production of pig embryos.

The routine maturation, fertilization, and development of pig embryos in vitro has only recently been achieved. Many of the conditions for in vitro production of embryos have been undefined and thus difficult to replicate. The major problems of in vitro production of pig embryos have included maturation of oocytes, both nuclear and cytoplasmic, and development from one-cell to blastocyst. While these barriers have been at least partially overcome there is still a significant problem with polyspermy. Nevertheless, numerous offspring have been produced from in vitro maturation, in vitro fertilization, followed by a brief culture prior to embryo transfer. Here is provided a review of the literature encompassing the current status of the in vitro production of pig embryos.

Effects of media NaCl concentration and osmolarity on the culture of early-stage porcine embryos and the viability of embryos cultured in a selected superior medium.

One- and 2-cell porcine embryos were obtained from oviductal flushes and cultured for 96 hours in media with varied osmolarity that resulted from alterations in NaCl and sorbitol content. The viability of experimental embryos cultured to advanced stages was determined by comparison with that of the controls, noncultured embryos transferred to recipient gilts. The data suggest that variation in embryonic development in the experimental media is related to the NaCl concentration rather than to osmolarity. Increased NaCl concentration impairs development of the embryos to the advanced morula/blastocyst stages (P<0.001). There was no difference in the pregnancy rate between the recipients of cultured (45%) and noncultured (57%) embryos on Day 25. There was, however, a higher embryonic survival rate (P<0.05) within the control gilts.

Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes.

The temporal progression of meiotic and cytoplasmic maturation of pig oocytes cultured in a medium supplemented with 0.4% polyvinylalcohol (PVA), 10% fetal calf serum (FCS), 10% newborn piglet serum (NPS), 10% porcine follicular fluid (PFF) or 10% porcine seminal fluid (PSF) was examined after 20, 30, 40 and 50 hours of culture. There were no differences in germinal vesicle breakdown (GVBD) among FCS and NPS supplements. After 20 hours of culture, the frequency for GVBD was higher (P < 0.05) in FCS and NPS (54% and 52%, respectively) than in PVA (32%) and PFF (33%) culture media but were not different at 40 and 50 hours of culture. Supplementation with PSF resulted in a rapid chromosome condensation of pig oocytes after 20 hours culture, but all GVBD oocytes stopped developing at the condensed germinal vesicle stage. Oocytes were not penetrated by spermatozoa when inseminated following 20 hours of culture, while high penetration (87 to 100%) and polyspermy rates (86 to 100%) were consistently obtained in all the supplement groups when inseminated after 30, 40 or 50 hours of culture. Male pronuclear formation rates at 10 to 12 hours after insemination, following a 50-hour culture period in FCS and NPS, were 28 and 28%, respectively, in comparison with 54% in PVA and 59% in PFF. The results indicate that supplementing maturation media with serum such as FCS and NPS reduced the ability of pig oocytes to form a male pronucleus, and further suggest that the detrimental effects may be due to accelerated progression of maturation events.

The effects of zearalenone on reproduction in swine. II. The effect on puberty attainment and postweaning rebreeding performance.

Eighty-five prepuberal, crossbred gilts received, ad libitum, a diet containing 0 or 10 ppm purified zearalenone for 30 d beginning at 145 to 193 d of age. At the end of this period all gilts were placed on the control diet and exposed daily to a mature boar for 60 d. Within 3 to 5 d of zearalenone ingestion, gilts showed marked vulval swelling and reddening, which continued for the 30-d feeding period. Thereafter symptoms slowly subsided. Zearalenone treated gilts showed first estrus significantly later than controls (P<0.05), but the proportion of gilts showing estrus within 60 d of boar exposure was similar (P>0.05). The length of the first estrous cycle was not affected by the ingestion of zearalenone before puberty (P>0.05). In a second study, 65 multiparous, crossbred sows were full-fed twice daily a ration containing 0 or 10 ppm of purified zearalenone beginning 14 d before weaning. Postweaning, all sows were fed the control diet, were checked for estrus daily, and inseminated at the first postweaning estrus. Neither sows nor gilts from their litters exhibited signs of hyperestrogenism during treatment. Weaning to estrus interval was significantly extended in zearalenone treated sows (P<0.05), but all other variables of fertility assessed were similar. These data suggest that zearalenone ingestion before puberty delays the stimulation of puberty associated with boar exposure, but does not affect subsequent cyclicity if zearalenone is removed from the ration. Similarly, zearalenone ingestion during lactation delays the return to estrus after weaning, but does not affect subsequent fertility when removed from the ration at weaning.

Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro.

The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

The effects of zearalenone on reproduction in swine. I. The relationship between ingested zearalenone dose and anestrus in non-pregnant, sexually mature gilts.

Ninety-nine sexually mature, non-pregnant gilts were checked for estrus daily with a mature boar and then allocated at estrus (D O) to receive 2 kg/d of a diet containing 0, 1, 5 or 10 ppm purified zearalenone between D 5 and 20 of the estrous cycle during two seasons of the year (winter and summer). None of the gilts exhibited any visual signs of "hyperestrogenism" and there was no effect of season on interestrous interval (P>0.05). A significant effect of zearalenone dose on inter-estrous interval was detected (P<0.001). Gilts receiving 0 or 1 ppm had similar inter-estrous intervals (21.0+/-0.3 and 21.5+/-0.8 d, respectively) whereas gilts receiving 5 and 10 ppm had extended cycles (29.2+/-2.9 and 32.7+/-3.3 d, respectively). Plasma progesterone concentrations at D 19 to 21 were higher in gilts with extended cycles (P<0.001) and corpora lutea (CL) were present at laparotomy. Some 86% of these retained CL underwent spontaneous regression resulting in the onset of estrus within the next 30 d. Fecal zearalenone concentrations rose during ingestion of contaminated diets and declined to pretreatment values within 2 d (1 ppm) to 8 d (10 ppm) of the cessation of treatment. These data show that feeding zearalenone at concentrations of 5 to 10 ppm from D 5 to 20 of the estrous cycle causes luteal maintenance and extended inter-estrous intervals. Spontaneous regression of these CL usually occurs within 30 d after zearalenone is removed from the diet. Fecal zearalenone analysis does not appear to be an effective method for determining prior exposure to zearalenone when carried out more than a few days following the last ingestion of zearalenone.

Parturition control in sows with a prostaglandin analogue (alfaprostol).

The objective of this study was to determine the dose of alfaprostol (18, 19, 20-trinor-17-cyclohexyl-13, 14 didehydro-PGF(2)alpha-methylester) and the day of administration most effective in inducing sows to farrow during normal working hours (0700 to 1700). One-hundred forty multiparous crossbred sows, taken from a herd whose mean gestation length was 114.3 days, were assigned to one of five treatment groups: 1) control vehicle-propylene glycol, 2) 0.5 mg alfaprostol (AP). 3) 1 mg AP, 4) 2 mg AP and 5) 3 mg AP. Sows received an intramuscular injection of AP between 0800 and 0830 on either day 111, 112 or 113 of gestation. Parameters studied included interval from injection to birth of first pig, farrowing interval, total number of pigs born, number born alive, average birth weight, percent stillborn, interval from weaning to next estrus and number born alive next litter. The mean intervals from injection to the birth of the first pig were 55.2 +/- 7.1; 41.1 +/- 5.1; 29.6 +/- 4.0; 24.3 +/- 1.1; 24.8 +/- 0.9 h for groups 1, 2, 3, 4 and 5, respectively (P<0.05). The percentage of sows that farrowed during normal working hours (23 to 33 h after injection) for groups 1, 2, 3, 4 and 5 were 15, 53, 60, 77 and 70%, respectively. Average birth weights (kg) for treatment groups 1, 2, 3, 4 and 5 were 1.54 +/- 0.05; 1.65 +/- 0.05; 1.59 +/- 0.05; 1.54 +/- 0.05 and 1.42 +/- 0.05, respectively (P<0.05). Mean differences in total number of pigs born and number born alive were also statistically significant (P<0.05) but stillbirth rate was not different (P>0.05) among treatment groups. These results indicate that a single injection of 1, 2 or 3 mg of alfaprostol will successfully induce parturition the following day in a majority of the treated sows.

Developmental ability of porcine oocytes matured and fertilized in vitro.

The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42 71 ) and with monospermic penetration and male and female pronuclei (32%, 23 71 ) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18 71 and 8%, 6 71 , respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28 288 and 13%, 41 318 , respectively) were also higher (P < 0.05) than in controls (2%, 6 284 and 6%, 16 248 , respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9 288 in PFF-treated group and 2%, 6 284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3 100 ) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

Embryonic survival in the uterus of ewes inseminated at the uterotubal junction on Day 32 post partum.

Experiments were conducted to determine 1) if pregnancy initiated on Day 32 post partum would be maintained until lambing, 2) if there is a difference in the ability of the previously gravid or nongravid uterine horn to maintain pregnancy, and 3) if season has an effect on embryo loss. Estrus was induced in ewes on Day 32 post partum. At estrus, ewes were inseminated surgically at the uterotubal junction and assigned to the following groups: 1) inseminated at estrus and laparotomized on Day 3 to collect embryos for determination of fertilization rate (C), 2) inseminated in the previously gravid uterine horn (PG), 3) inseminated in the previously nongravid uterine horn (NG), and 4) inseminated when both horns were previously gravid (BG). Ewes pregnant in the PG, NG and BG groups were allowed to lamb. Conception rate in Group C at embryo collection was 70%. Embryo loss, based on concentrations of progesterone at Day 18 post insemination, was 43, 19 and 18% in the BG, NG and PG group, respectively. The high embryo loss in Group BG occurred only during the breeding season. Only 24% of the ewes that had been inseminated lambed. This was due to the prepartum loss of embryos and fetuses (47, 48 and 33% in Group BG, NG and PG, respectively. In conclusion, the detrimental effects of the uterus on embryo survival was evident within 18 d post insemination in Group BG (breeding season), and embryo loss prior to lambing was high in all the treatment groups (both seasons).

Plasma prolactin concentration in gilts reared in confinement.

Plasma prolactin concentrations were determined in confinement-reared gilts, which were subsequently exposed to boars and/or relocated to pasture lots. At an average age of 181 days, an indwelling vascular cannula was surgically implanted, and 28 gilts were assigned randomly to either control (remained in confinement) or treatment (relocated to pasture) groups. Nine of the gilts in each group were exposed to a mature boar twice daily. Furthermore, the experiment was divided between April (n=17 gilts) and September (11 gilts). Blood samples were drawn at the time of surgical implantation and twice daily thereafter. Plasma concentrations of prolactin were quantified by validated radio-immunoassay procedures. Samples which were collected at surgery had significantly more prolactin than post-surgical samples in 25 of 28 gilts. The overall average for the post-surgical concentration of prolactin was 2.7 ng/ml, and there was no effect of continued confinement versus relocation to pasture lots. There was a tendency for boar exposure to reduce prolactin levels in relocated gilts but not in control gilts. There was also a tendency for plasma prolactin to be greater in September than in April, especially in control gilts. Overall these results indicate that plasma prolactin levels do not reflect the delay of puberty in confinement-reared gilts or the induction of puberty caused by relocation to pasture lots.

GnRH induced LH release in suckled beef cows. 1. The effects of days postpartum and estradiol-17beta concentrations on the release of LH following administration of GnRH.

Twenty suckled CharloixxHereford beef cows (5 cows/group) were assigned at random to receive 100 microg GnRH (IM) at either 2 to 3, 7 to 8, 15 to 16, or 31 to 32 days postpartum, Groups 1 through 4, respectively. Blood samples for hormone determinations were collected at time 0 (pre-GnRH), every half hr for 3 hr, and at 4.0 hr and 6.0 hr post-GnRH. Mean plasma LH, estradiol-17beta, or progesterone concentrations were not different among groups prior to GnRH. Plasma LH increased (P<.05) following GnRH in Groups 2, 3 and 4, but not in Group 1. Peak GnRH induced LH release was greater (P<.05) in Groups 3 and 4 than in Groups 1 or 2. Correlation coefficients between days postpartum and peak LH release (r=.72), and estradiol-17beta concentrations and time of LH peak (r=-.42) were significant (P<.05). These data indicate that LH release in response to GnRH, in suckled beef cows is not fully restored until 15 to 16 days postpartum.