Dihydrotestosterone influenced numbers of healthy follicles and follicular amounts of LH receptor mRNA during the follicular phase of the estrous cycle in gilts.

The present experiments were conducted to determine androgenic effects on numbers, health, and amounts of gonadotropin receptor mRNA in late developing follicles of gilts. Gilts (n=5 per group) received daily injections of one of the following treatments on days 13-16 or days 13-18 of the estrous cycle: corn oil, 5alpha-dihydrotestosterone (DHT, 10 mg), flutamide (1.5 g, an androgen receptor inhibitor), DHT (10 mg) plus flutamide (1.5 g), testosterone (10 mg), and testosterone (10 mg) plus flutamide (1.5 g). Ovarian follicles > or =5 mm in diameter were evaluated on day 17 or 19, 24 h after receiving the last treatment dose. Follicles were classified as healthy (H), moderately atretic (MA), or very atretic (VA). Treatment with DHT increased (P<0.05) the numbers of H follicles relative to control gilts on days 17 and 19. DHT administration from days 13 to 16 diminished (P<0.05) the amounts of LH receptor (LHR) mRNA in H follicles from day 17 (relative amounts: 1.45+/-0.33 and 2.72+/-0.33 for DHT- and vehicle-treated gilts respectively). The effects of DHT on numbers of H follicles and LHR mRNA were not observed in gilts receiving DHT plus flutamide. Androgens did not influence numbers of MA, VA, and total follicles, or follicular estradiol-17beta concentrations and amounts of FSHR mRNA. Treating gilts with DHT during follicular recruitment and selection did not induce changes in the numbers of total follicles > or =5 mm, but rather increased the numbers of healthy follicles in this follicular population in association with decreased amounts of LHR mRNA.

Androgens in female pig reproduction: actions mediated by the androgen receptor.

Androgens have potential actions in almost all the organs of males and females. In females, most organs contain some tissues with cells that have androgen receptors. Androgens can regulate cellular functions by binding to androgen receptors or be converted to other hormones. For example, testosterone can bind to the androgen receptor or be aromatised to oestradiol. Treating animals with testosterone, therefore, might elicit some androgenic and oestrogenic effects. Alternatively, testosterone can be converted to other androgens, which in turn, have more or less affinity with the androgen receptor and these new metabolites may or may not be aromatised to oestrogens. This review will highlight the roles of androgens in female mammals other than those as a substrate for oestrogen, with particular emphasis on the actions of the androgen receptors in uteri and ovaries of pigs. Utilising small dosages of an androgen receptor agonist, DHT (5alpha-dihydrotestosterone) we have observed that some uterine functions were inhibited while ovarian follicular development was augmented. These inhibitory and stimulatory effects of androgen therapy on reproductive organs can potentially be balanced to enhance ovulation rate and litter size in gilts and sows. Perhaps after future experimentation, new uses of androgens or anti-androgens could improve additional aspects of sow performance not presently under consideration.

Intracellular adenosine triphosphate and glutathione concentrations in oocytes from first estrous, multi-estrous, and testosterone-treated gilts.

Cytoplasmic maturation refers to a variety of cellular changes that must occur in the oocyte in order to progress through subsequent fertilization and embryonic development. Intracellular concentrations of ATP (ATPi) or glutathione (GSHi), indicative of metabolic activity or the ability of the oocyte to form a male pronucleus and cope with cellular stress, respectively, have been used as markers of cytoplasmic maturation in vitro. In the current study, our objective was to determine if concentrations of ATPi and GSHi in oocytes recovered from three groups of gilts were associated with known differences in developmental competence within these populations. In vivo matured oocytes were surgically recovered 36-38 h after the onset of estrus from first estrous gilts, multi-estrous gilts, and multi-estrous gilts receiving testosterone (1mg/2 ml per day; day 13 to estrus, day 0=day of estrus). Concentrations of ATPi and GSHi were determined using a bioluminescent somatic cell assay kit (luciferin-luciferase reaction) and the dithiobisnitrobenzoic acid (DTNB)-glutathione reductase recycling reaction, respectively. There were no differences (P>0.05) between ATPi concentrations in oocytes from the three groups (1.52 +/- 0.10, 1.51 +/- 0.11, 1.56 +/- 0.11pmol per oocyte). In contrast, oocytes from multi-estrous gilts had higher (P<0.05) concentrations of GSHi (31.53 +/- 1.66 to 33.67 +/- 2.30 pmol per oocyte) than oocytes from first estrous gilts (25.07 +/- 0.82). Administration of testosterone did not affect (P>0.05) GSHi concentrations in oocytes from multi-estrous gilts. Differences in developmental potential between the three groups of gilts were apparently not due to different concentrations of ATPi. However, GSHi concentrations were higher in oocytes from multi-estrous gilts, suggesting that reduced developmental potential of oocytes from first-estrus gilts may be related to insufficient amounts of GSHi. The beneficial effect of exogenous testosterone on the percentage of embryos surviving early gestation does not appear to be due to increased GSHi. Of the numerous potential markers of developmental potential, two were examined in the current study, and GSHi appeared to be useful for assessing porcine oocytes.

Distribution and changes in amounts of the androgen receptor in the pig uterus during the estrous cycle, early pregnancy and after treatment with sex steroids.

Two experiments were performed to examine the expression of the androgen receptor (AR) gene in the pig uterus. In experiment 1, immunohistochemistry (IHC) was used to determine the distribution of the AR in uterine tIssue of pigs when collected at the first day of estrus (day 0) and the mid-luteal phase (day 12) of the estrous cycle, or early pregnancy (day 12, n=4 gilts per group). In experiment 2, AR immunostaining and AR mRNA in uterine tIssue were compared among ovariectomized gilts (n=4 per group) following treatment for 4 days with daily injections of: (1) progesterone (2 mg/kg bodyweight (BW)), (2) estradiol-17beta (E(2,) 2 micro g/kg BW), (3) E(2) plus progesterone (same dosages as 1 and 2 combined), (4) 5alpha-dihydrotestosterone (DHT, 7 micro g/kg BW), or (5) vehicle (corn oil). Data were analyzed using ANOVA. In experiment 1, nuclear staining for AR in luminal and glandular epithelia was strong and did not differ in intensity between the two locations. Immunostaining of AR in the myometrium was less (P<0.001) intense than in the luminal and glandular epithelia. Nuclei of stromal cells contained AR immunostaining that varied in intensity from strong (mainly in subepithelial stroma) to weak or no staining. Stages of the estrous cycle or early pregnancy did not influence AR immunostaining in the endometrial epithelia and myometrium. In experiment 2, immunostaining of AR in glandular and luminal epithelia and myometrium of ovariectomized gilts treated with vehicle or DHT was less (P<0.05) than in gilts treated with E(2), progesterone, or E(2) plus progesterone. Immunostaining of AR did not differ between ovariectomized gilts treated with vehicle or DHT, or between gilts treated with E(2), progesterone, or E(2) plus progesterone. In both experiments, intensity of AR immunostaining was greater in glandular epithelium located at the adluminal region compared with glandular epithelium located at the basal region of the endometrium. Competitive reverse-transcription PCR (RT-PCR) indicated a stimulatory effect (P<0.01) of E(2) on amounts of AR mRNA in whole endometrium. This increase in AR mRNA after E(2) treatment was not detected when E(2) was combined with progesterone. Endometrial AR mRNA was not influenced by DHT or progesterone relative to vehicle-treated gilts. In conclusion, immunoreactive AR is mainly present in luminal and glandular epithelia of the pig uterus and to a lesser extent in the myometrium, and does not change significantly during the estrous cycle or early pregnancy. Expression of the AR gene in the pig endometrium and myometrium appears to be regulated by E(2) and progesterone.

Exposure to androgens during in vitro maturation does not affect the developmental potential of porcine oocytes.

Administration of exogenous androgens to pigs during the period of follicular development has been shown to positively affect ovulation rate and embryonic survival. The mechanisms of these actions are not known, but may include direct effects of androgens on the cumulus-oocyte complex (COC). The objective of this experiment was to assess the effects on embryonic development in vitro of exposure of COC to 0.26 and 2.6 microM testosterone (T) or dihydrotestosterone (DHT) during IVM. For IVM, COC were cultured for 44-46 h in protein-free tissue culture medium (TCM) 199 containing 10 IU/ml hCG and eCG and 10 ng/ml EGF. Oocytes were then stripped of cumulus cells, coincubated with 1 x 10(5) sperm/ml in modified TALP for 6 h, and cultured for 8 days in NCSU23. The proportions of oocytes that cleaved (Day 2) or developed to the morula (Day 6) or blastocyst (Day 6-8) stage were not different (P > 0.20) between oocytes exposed to androgens and oocytes not exposed to androgens. These results indicate that exposure to androgens during IVM does not affect the ability of oocytes to cleave or develop up to the blastocyst stage in vitro.

Androgen receptor and follicle-stimulating hormone receptor in the pig ovary during the follicular phase of the estrous cycle.

Follicle-stimulating hormone (FSH) is an important regulator of follicular development. Some effects of FSH on ovarian follicles might be enhanced by androgens. The main objectives of the present study were to examine expression of the androgen receptor (AR) and FSH receptor (FSHR) in late developing follicles in pigs. Ovaries were collected from gilts on days 13, 15, 17, and 19 of the estrous cycle (day 0 = first day of estrus, n = 4 gilts/day), a period coincident with the follicular phase. One ovary was processed for immunohistochemistry (IHC) of AR. Samples of surface wall from the largest follicles (4-5 per gilt) were dissected from the other ovary, pooled and processed for determination of AR and FSHR mRNAs using reverse transcription-polymerase chain reaction (RT-PCR). Intense AR immunostaining was present in nuclei of granulosa cells of preantral and antral follicles. AR immunoreactivity was also present in the nuclei of oocytes. Weak staining for AR was observed in cells of the theca interna, ovarian surface epithelium, and in most cells of the ovarian stroma. Relative amounts of immunoreactive AR in granulosa cells of late developing follicles, or small antral follicles (< 2 mm), did not differ between days 13, 15, 17, and 19. However, amounts of AR in granulosa cells of small antral follicles was greater (P < 0.05) than in the largest follicles present in the same ovary. The relative amounts of AR mRNA in tissue from the largest follicles on days 13, 15, 17, and 19 did not differ; however, amounts of FSHR mRNA in the same follicles were not different between days 13, 15, and 17, but decreased (P < 0.05) by day 19. Results indicate that during the follicular phase in gilts, the AR protein is mainly present in granulosa cells. Relative amounts of AR protein in granulosa cells and mRNA in walls of late developing follicles did not significantly change from day 13 to 19; however, amounts of FSHR mRNA decreased in preovulatory follicles by day 19 of the estrous cycle.

Increased ovulation rate in gilts treated with dihydrotestosterone.

Treatment with testosterone increases ovulation rate in pigs. The present study was conducted to examine the effects of 5alpha-dihydrotestosterone (DHT), a non-aromatizable androgen receptor ligand, on ovulation rate and amounts of androgen receptor and FSH receptor mRNAs in postpubertal gilts. In Expt 1, ovulation rate in response to daily i.m. injections of 0, 6, 60 or 600 microg DHT kg(-1) body weight from day 13 of the oestrous cycle (day 0 = day 1 of oestrus) to the following oestrus increased with each dose of DHT (P < 0.05). The mean increase in number of corpora lutea ranged from approximately three to 17 over the three dosages of DHT. In Expt 2, gilts treated daily with 60 microg DHT kg(-1) body weight during the early follicular phase (from day 13 to day 16), coincident with follicular recruitment, or the late follicular phase (day 17 to oestrus), had higher (P < 0.05) rates of ovulation compared with gilts that received vehicle, and were not different from gilts treated with DHT from day 13 to oestrus. Percentage recovery of day 3 embryos was not altered when gilts were treated from day 13 to day 16 or from day 17 to oestrus; however, treatment of gilts with DHT from day 13 to oestrus decreased recovery of day 3 (Expt 1) or day 11 (Expt 2) conceptuses. Daily administration of 6 microg DHT kg(-1) body weight to gilts from day 13 of the oestrous cycle to the following oestrus (Expt 3) did not affect the relative amounts of androgen receptor mRNA, but increased (P < 0.05) the amounts of FSH receptor mRNA in preovulatory follicles as determined by RT-PCR. The results of these experiments indicate that androgens may regulate ovulation rate in gilts. One of the roles of androgens might be regulation of the amounts of FSH receptor mRNA in ovarian follicles.

Control of anthelmintic resistant endoparasites in a commercial sheep flock through parasite community replacement.

An effort was undertaken to replace a community of sheep endoparasites that had been classified as resistant to levamisole and albendazole with a community of more susceptible parasites using a dilution approach that could be integrated into the management of a commercial flock. For this study, pastures on this sheep farm were divided into two areas: north and south. Strategically timed anthelmintic treatments combined with pasture management reduced to nondetectable levels the endemic community of anthelmintic resistant parasites in this flock and on these pastures by early summer. A group of 102 ewes, lambs, and rams were experimentally infected with third stage larvae from the more susceptible community of parasites. These sheep then seeded the south pastures with the new parasite community, while sheep on the north pastures maintained the endemic resistant community. Despite its insensitivity as a technique for detecting anthelmintic resistance, fecal egg count reduction tests at the end of the grazing season indicated that the more susceptible parasites were present on the south pastures while resistant parasites were present on the north. The following grazing season, similar protocols were used to introduce the more susceptible parasites onto the north pastures. At the end of the grazing season, fecal egg count reduction tests indicated that the new community of parasites had become established on both groups of pastures of the farm.

Estrogen receptor beta in the sheep ovary during the estrous cycle and early pregnancy.

Objectives were to sequence and examine the expression of the estrogen receptor beta (ERbeta) in the sheep ovary. The sequence of the ovine ERbeta (oERbeta) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERbeta contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERbeta. In addition, an oERbeta isoform having a 139-base pair deletion (oERbeta1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERbeta protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERbeta protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERbeta was detected in the theca interna. Relative steady-state amounts of oERbeta mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERbeta mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERbeta to oERbeta1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERbeta1. Results indicate that the oERbeta is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERbeta might regulate estrogen actions during early CL development in sheep.

Evidence for expression of estrogen receptor cofactor messenger ribonucleic acid in the ovary and uterus of domesticated animals (sheep, cow and pig).

Expression levels of estrogen receptor cofactors (coactivators or corepressors) in specific tissue compartments and cells are thought to influence the expression of estrogen responsive genes and thereby influence overall hormonal responsiveness of target tissues. To date, the presence of cofactors has been reported in tissues from humans, rats and mice. We analyzed the presence and distribution of messenger ribonucleic acids (mRNAs) encoding several transcriptional cofactors in the ovary and uterus of three domestic animal species, the sheep, cow and pig. Northern analysis for cofactors SRC-1, GRIP1, RAC3, p300, RIP140, and SPA showed expression in ovaries from all three species. In addition, lower expression of SRC-1, GRIP1, RAC3, p300, and RIP140 mRNAs was observed during the luteal phase (day 10-12 of the estrous cycle) than at estrus (day 0); however, SPA transcript levels remained unchanged. We then examined expression of mRNAs for changing (SRC-1, RIP140) and constitutively expressed (SPA) cofactors in ovine ovaries. SRC-1 and RIP140 transcripts in corpus luteum were lower compared to the surrounding ovarian tissue. SPA mRNA expression, however, was similar in corpus luteum and surrounding tissues. To determine which ovarian cell types express SRC-1, RIP140, and SPA, in situ hybridization was performed on sheep ovaries. Silver grains corresponding to these cofactors were seen in ovarian granulosa, theca and stromal cells, but appeared to be most abundant in the granulosa cells. Expression of SRC-1 and RIP140 in corpus luteum, however, was reduced compared to expression in follicular cells. Finally, we examined cofactor expression in ovine, bovine, and porcine uterus. Northern blot analysis for SRC-1, GRIP1, RAC3, p300, and RIP140 mRNAs showed higher expression in extracts of the endometrium compared to whole uterus. We provide the first evidence for the presence of estrogen receptor cofactor mRNAs in the ovary and uterus of three domestic animal species. We suggest that coactivators are conserved among species and associated with hormonal responsiveness of reproductive tract tissues in sheep, cow and pig.

Depletion of vitamin C from pig corpora lutea by prostaglandin F2 alpha-induced secretion of the vitamin.

The luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) are thought to be mediated in part by the promotion of an increasingly oxidative cellular environment. Loss of antioxidants is one mechanism by which PGF2 alpha might induce or exacerbate oxidative damage within the corpus luteum. This study was performed to establish whether depletion of vitamin C is an acute effect of PGF2 alpha on the pig corpus luteum and to gain insight into the mechanism of luteal vitamin C loss at luteolysis. Gilts (n = 4) were anaesthetized and both utero-ovarian veins and an ear vein were catheterized. Each corpus luteum on the treated ovary received an intraluteal injection of PGF2 alpha (1 microgram) followed by a sustained release implant containing 100 micrograms of the prostaglandin. The other ovary served as the control and each corpus luteum received corresponding volumes of injection vehicle and blank implant. Blood was collected from the ear vein and both utero-ovarian veins every 15 min beginning 15 min before the onset of treatment. Collection of blood stopped when animals were ovariectomized and corpora lutea were collected at 2 h after treatment. Progesterone and vitamin C (ascorbate) concentrations were measured in tissue and plasma samples. PGF2 alpha-treated luteal tissue had similar progesterone, but significantly lower ascorbate, concentrations when compared with control corpora lutea. PGF2 alpha treatment resulted in a rapid and sustained increase in plasma ascorbate within the treatment-side utero-ovarian vein, while the control utero-ovarian vein and ear vein showed little change in plasma ascorbate during the experimental period. No effect of PGF2 alpha on plasma progesterone was evident. This finding suggests that PGF2 alpha depletes the pig corpus luteum of vitamin C by inducing secretion of the vitamin into the bloodstream. Further studies are necessary to determine whether the depletion of vitamin C that is induced by PGF2 alpha contributes to the demise of the pig corpus luteum.

Cyclic and maturation-dependent regulation of follicle-stimulating hormone receptor and luteinizing hormone receptor messenger ribonucleic acid expression in the porcine ovary.

We sought to characterize the ovarian expression of mRNAs for the receptors for FSH (FSHr) and LH (LHr) at the different stages of the estrous cycle of the pig and to localize the receptor mRNAs to individual cell types in follicles and corpora lutea as a function of their developmental status. Northern blot analyses indicated that multiple FSHr and LHr mRNA transcripts occur on Days 0, 4, 7, 12, and 16 of the estrous cycle. In situ hybridization analyses revealed considerable cyclic variation in expression among small, medium, and large follicles. In small follicles of both immature prepubertal ovaries and mature adult ovaries at all days of the cycle studied, FSHr mRNA expression was strongly positive and was spatially restricted to granulosa cells. FSHr mRNA expression was strongly positive in granulosa cells of medium follicles but had declined in granulosa cells of mature follicles on Day 0. LHr expression in small follicles, in contrast, was spatially restricted to theca cells and was estrous cycle-dependent. LHr expression in theca cells was weakly positive in small follicles on Days 12, 0, and 4, was strongly positive in small follicles on Day 7 and in medium follicles on Days 12 and 16, and had declined in large mature follicles on Day 0. LHr expression in granulosa cells was negative in small follicles on Days 4 and 7, weakly positive in medium follicles on Days 12 and 16, and positive in large follicles on Day 0. In degenerating follicles, LHr mRNA was expressed weakly in the theca cells, and FSHr mRNA expression in granulosa cells was absent. In the corpus luteum, FSHr mRNA was not expressed, but LHr mRNA was expressed in some cells on Days 4 and 7, was expressed maximally on Day 12, and had declined partially by Day 16. These studies indicate that there is maturation-dependent regulation of FSHr and LHr expression in the adult porcine ovary. FSHr mRNA content decreases in parallel with FSHr protein content as follicles increase in size.

Effects of dietary selenium and vitamin E on boar performance and tissue responses, semen quality, and subsequent fertilization rates in mature gilts.

Three experiments involving 192 crossbred boars evaluated the effects of dietary Se (0 or .5 ppm) and vitamin E (0 or 220 IU/kg) on growth, tissue Se, and alpha-tocopherol concentrations, and on semen quality and its subsequent effect on fertilization rate in mature gilts. Diets formulated used torula yeast and dextrose or cornstarch as the basal feedstuffs and were provided from weaning through sexual maturity. The basal diets averaged .063 ppm Se and 3.46 mg alpha-tocopherol/kg diet. Experiment 1 was a 2 x 2 factorial and conducted as a randomized complete block design in six replicates. Boars were allotted at weaning (initial BW 7.7 kg) with growth and feed performance determined to 145 kg BW. Five boars were killed at weaning and three from each treatment group at periodic intervals to 145 kg BW. Serum and tissue Se and alpha-tocopherol concentration and glutathione peroxidase (GSH-Px) activity were subsequently determined. No performance benefit from either nutrient was demonstrated. Tissue (serum, liver, and testis) GSH-Px activity and Se and alpha-tocopherol concentrations were higher (P < .01) at each period when that respective nutrient fortified the diet. Testis GSH-Px activity increased from weaning to 145 kg BW even when Se was not added to the diet. Experiment 2 was conducted after training three boars from each treatment group of Exp. 1 for semen collection. From 9 mo of age and for a 16-wk period, semen was collected three times weekly and the volume, sperm concentration, motility, and percentage of normal and abnormal sperm were determined. Boars fed either the nonfortified Se or vitamin E diets had sperm with lower motilities (P < .01) and a higher percentage of sperm cells with bent and shoehook tails (P < .01). Diets low in added Se seemed to have a greater detrimental effect on the percentage of motile and abnormal sperm than diets inadequate in vitamin E. Sperm cells had a high concentration of Se and alpha-tocopherol, and a high GSH-Px activity. Experiment 3 was conducted using the boars from Exp. 2; 34 mature gilts were inseminated at 12 and 24 h after estrus. Gilts were killed 5 to 7 d postcoitum and the reproductive tracts were recovered. The semen from boars fed the nonfortified Se diet had a lower fertilization rate of oocytes with fewer accessory sperm penetrating the zona pellucida. The results from these experiments indicate that dietary Se and vitamin E can affect boar semen quality, but the greater effect seemed to be from Se.

Short-term effects of exogenous estradiol-17 beta on blastocyst development during the period of elongation in swine.

Objectives were to examine the effects of a single dose (4 mg) of estradiol-17 beta (E2) on blastocyst development around the period of elongation. Proestrus gilts were induced to ovulate with 750 IU of hCG and were mated before ovulation (normal mating, 24 to 32 h post-hCG) or after ovulation had begun (delayed mating, 43 h post-hCG). This difference in time of mating has been demonstrated to result in approximately a 7-h difference in time of blastocyst elongation. Normally and delay-mated gilts were ovariohysterectomized at 278 h post-hCG or injected with E2 or vehicle (corn oil) at 278 h and then ovariohysterectomized at 290 h post-hCG (five or six gilts per group). Blastocyst size was measured and concentrations of E2, retinol, uteroferrin, insulin-like growth factor-I (IGF-I), uterine plasmin/trypsin inhibitor (UPTI) and protein in uterine flushings were quantified. Blastocyst size and components of uterine flushings did not differ (P > 0.05) between normally and delay-mated gilts at 278 h post-hCG. However, at 290 h post-hCG, normally mated gilts had larger (P < 0.01) blastocysts (small spheres to filamentous) and their flushings tended to contain less (P < 0.07) amounts of retinol than those of delay-mated gilts whose blastocysts ranged from small spheres to ovoidals. Normally mated gilts receiving E2 at 278 h had smaller (P < 0.01) blastocysts and less (P < 0.05) amounts of retinol at 290 h post-hCG than gilts receiving vehicle. Conversely, delay-mated gilts treated with E2 or vehicle did not differ (P > 0.05) in blastocyst size and amounts of components of uterine flushings at 290 h post-hCG. Normally mated gilts treated with vehicle had litters in the process of elongating at 290 h post-hCG. Mean blastocyst size (P < 0.001) and amounts of components of uterine flushings (except for IGF-I) in these gilts were greater (P < 0.05, UPTI = 0.06) than in normally mated gilts at 278 h post-hCG, whose blastocysts were spherical. Among gilts not treated with E2 (278 h and 290 h pooled), mean blastocyst size was positively correlated (P < 0.05) with amounts of retinol, E2, uteroferrin and total protein. Results indicated that a single dose of E2 given before elongation altered blastocyst development depending on how close blastocysts were to onset of elongation at the time of E2 treatment.

Effect of the rate of progesterone decline at luteolysis on the ovulatory follicles and subsequent estrous cycle length in ewes.

Follicular and interestrous characteristics were examined in 34 ewes after experiencing either a rapid decline in plasma concentrations of progesterone at luteolysis [prostaglandin F2 alpha (PGF2 alpha)-induced] or a slow rate of decline, lasting over 72 h. All ewes were given PGF2 alpha on day 10 (day 0 = estrus). A slow rate of decline was established in 17 ewes by the intravenous infusion of progesterone initially at 72 ml h-1, delivering 4.5 mg progesterone h-1, then decreasing the infusion rate by 1 ml h-1 for the next three days. Seventeen additional ewes, predestined to experience a rapid decline in progesterone, were infused with vehicle. In Experiment 1, after infusion, ewes (6 ewes/group) were necropsied at the onset of estrus and follicle diameter was determined, follicular fluid was aspirated and the remaining follicular wall was microscopically examined to determine the number of granulosa cell layers. In Experiment 2, the interestrous interval, after infusion, was observed in both groups of ewes (11 ewes/group). Ewes experiencing a rapid rate of progesterone decline at luteolysis had no differences in follicle diameter nor follicular concentration of progesterone or estradiol but their ovulatory follicles contained fewer (P < 0.01) granulosa cell layers and the resulting estrous cycle was longer (P < 0.05) than ewes experiencing a slow rate of progesterone decline.

Administration of testosterone from day 13 of the estrous cycle to estrus increased the number of corpora lutea and conceptus survival in gilts.

The effects of exogenous androgens on the number of corporea lutea (CL) and conceptus survival were examined in crossbred gilts. In Exp. 1, gilts received 1 mg of testosterone per day from d 13 (d = 0 first day of estrus, n = 21) or d 16 until estrus (n = 23). Gilts in the vehicle group received corn oil (n = 20). Gilts were mated and on d 11.5 their concepti and CL were evaluated. In Exp. 2, conceptus survival was examined at the 4- to 8-cell, early blastocyst or hatching blastocyst stages for gilts given vehicle or 1 mg testosterone from d 13 (24 gilts per group). In Exp. 3, gilts received 1 mg of androstenedione (n = 20) or vehicle (n = 18) per day from 13 d to estrus and then were mated and evaluated on d 11.5. Results from Exp. 1 indicated that the number of CL was greater (P < .04) in gilts treated with testosterone from d 13 to estrus than in gilts receiving vehicle (16.4 vs 14.8, respectively). Similarly, the number (P < .01) and recovery rate (P < .04) of blastocysts were greater in gilts treated with testosterone from d 13 to estrus than in gilts treated with testosterone from d 16 to estrus in gilts receiving vehicle (number, 15.3 vs 12.8 or 12.8; recovery rate, 95 vs 87 or 86%, respectively). Gilts treated testosterone or vehicle did not exhibit differences (P > .05) in number of normal concepti at the 4- to 8-cell and hatching stages. However, prior treatment with testosterone delayed conceptus death; gilts treated with testosterone had more (P < .01) normal concepti at the intermediate stage (early blastocyst) than those treated with vehicle (treatment x embryo stage interaction, P < .05). In Exp. 3, androstenedione treatment did not influence (P > .10) the number of CL or the number and recovery rates of d-11.5 blastocysts. Treating gilts with testosterone from d 13 of the estrous cycle to the following estrus increased the number of CL and blastocyst survival, perhaps by improving some, as yet unknown, aspect(s) of oocyte quality.

Administration of testosterone during the follicular phase increased the number of corpora lutea in gilts.

The effects of 0 (vehicle), 1, 10, or 100 mg of testosterone, administered on d 17 and 18 of the estrous cycle (d 0 = 1st d of estrus), on the number of preovulatory follicles on d 19 or the number of corpora lutea (CL) and blastocysts on d 11 of the subsequent cycle were examined in 82 gilts. The mean number of preovulatory follicles increased (P < .01) in a dose-dependent manner. Likewise, gilts that received 1, 10, or 100 mg of testosterone had more (P < .05) CL than gilts treated with vehicle. The mean number of blastocysts increased (P = .06) in gilts receiving 1 mg of testosterone but decreased (P < .05) in gilts treated with 10 mg of testosterone compared with gilts receiving vehicle. Similarly, recovery rates of blastocysts decreased (P < .05) in gilts treated with 10 or 100 mg of testosterone relative to gilts administered 0 or 1 mg of testosterone. Plasma concentrations of testosterone and estradiol increased (P < .05) 2 h following administration of 1 mg of testosterone on d 17 of the estrous cycle. These results indicate that although the 10- and 100-mg dosages of testosterone were detrimental to blastocyst survival, the 1-mg dosage increased synthesis of estradiol and the number of CL and d-11 blastocysts.

Selective cloning of cDNA for secretory proteins of early embryos. Identification of a transiently expressed kunitz domain protein from preimplantation sheep trophoblast.

The preimplantation ovine conceptus transiently secretes several proteins, including a type I interferon (IFN-tau), that are likely involved in establishment of pregnancy. A method has been developed to identify proteins produced simultaneously with IFN-tau. An antiserum against total ovine conceptus secretory proteins, from which IFN-tau and proteins common to maternal uterine tract secretions had first been removed, was used to immunoscreen cDNA libraries created from mRNA of days 13 and 15 ovine conceptuses. This approach has allowed several unique cDNA to be identified, including one particularly abundant transcript for a novel member of the Kunitz family of serine protease inhibitors. This cDNA encodes a 265-amino acid protein with a 20-amino acid signal sequence. A 64-amino acid Kunitz domain occupies the carboxyl terminus. It is preceded by two similar repeats of 84 residues that bear no obvious similarity to any sequences present in the protein data banks. The protein present in conceptus secretions (M(r) of approximately 14,000) represents only the carboxyl terminus of the molecule. The mRNA for this putative proteinase inhibitor was confined to trophectoderm and was highly expressed for only a few days (approximately 13-18) of development. A similar transcript was detected during the days 17-21 period in cattle embryos. Despite their high expression, no proteinase-inhibitory activity can so far be ascribed to either the ovine or bovine proteins. The P1 residue, an asparagine, is not represented in any other known Kunitz inhibitors.

Effects of administration of human chorionic gonadotropin or progesterone before maternal recognition of pregnancy on blastocyst development and pregnancy in sheep.

A series of four experiments with 258 ewes was conducted to determine whether blastocyst size could be altered before normal luteolysis and, if so, how this affected fertility. In Exp. 1 and 2, nonmated and mated ewes, respectively, were treated with hCG (100 IU), progesterone (12 mg), or vehicle on d 11.5 (d 0 = onset of estrus). In Exp. 3 and 4, field trials were conducted to compare the effects of either hCG or progesterone treatment on d 11.5 on subsequent pregnancy rates. In Exp. 1, hCG transiently increased (P < .01) concentrations of progesterone and estradiol in plasma, whereas progesterone treatment increased only plasma progesterone. Neither hCG nor progesterone affected the duration of the estrous cycle. In Exp. 2, d-13 blastocysts were longer (3.5 +/- 1.6 vs .8 +/- .5 cm; Mean +/- SE; P < .05), and concentrations of protein and interferon tau (IFN tau) in uterine flushings were greater (10.7 vs 1.2 micrograms; P < .05) in hCG than in vehicle-treated ewes. Progesterone treatment did not affect blastocyst development. In Exp. 3, pregnancy rates tended to be greater (P < .10) in ewes given hCG than in those given vehicle (44/47; 94% vs 40/48; 83%); however, administration of progesterone in Exp. 4 had no effect on pregnancy rates (P < .14; 41/45; 91% vs 37/46; 80%; control ewes). These results indicate that treatment with hCG on d 11.5 stimulated uterine secretions and conceptus growth sufficiently to influence pregnancy rates.

Effects of progesterone pretreatment on fertility of gilts mated at an induced pubertal estrus.

The effects of progesterone (100 mg/d, im) on pubertal fertility were examined in 247 gilts over 3 experiments. In the first experiment, 128 gilts were exposed to progesterone for 0, 2, 4 or 8 d before receiving PMSG (750 IU) 1 d later. The number of large (>4mm) follicles or corpora lutea (CL) were determined on the day of PMSG injection, Day 0 (onset of estrus), Day 1 or Day 10 (n=8). In the second experiment, embryonic survival was observed in 68 gilts after induction of estrus with PG600 (400 IU PMSG, 200 IU hCG). Vehicle or progesterone was previously administered for 2 d to these gilts, and they were allowed 1, 2, or 3 d between the last progesterone injection and PG600. In Experiment 3, a field trial was conducted in which 51 gilts received vehicle or progesterone for 2 d, followed by a 3-d interval before injection of PG600 to induce estrus. The gilts were allowed to farrow. Treatment with progesterone 1 d before PMSG increased (P<0.05) the number and size of preovulatory follicles and increased (P<0.05) the number of corpora lutea. However, the percentage of gilts pregnant by Day 10, the number of embryos recovered per gilt and embryonic survival were reduced (P<0.05) with progesterone pretreatment. Utilizing a smaller dose of PMSG (750 vs 400 IU) with PG600 negated the effects of progesterone pretreatment on ovulation rate. When the interval between progesterone treatment and PG600 was lengthened to 3 d embryonic survival to Day 30 improved but was similar to that of the vehicle/PG600 treated gilts. Fertility, as defined as conception rate and litter size, was similar between gilts exposed to vehicle or progesterone. These results indicate that pretreatment with progesterone up to the day before PMSG might improve follicular development and ovulation rate at the pubertal estrus with a dose of 750 IU of PMSG but not with the 400 IU (PG600). Reducing the dose of PMSG to 400 IU and allowing for 3 d between progesterone and gonadotropin treatment reduced the incidence of uterine infections but resulted in a fertility rate similar to that of gilts receiving PG600 alone.