Control of interferon-tau secretion by in vitro-derived bovine blastocysts during extended culture and outgrowth formation.

A series of experiments was conducted to examine the pattern of interferon-tau (IFN-tau) secretion by bovine blastocysts during extended culture in vitro. In the first experiment, blastocysts were cultured individually for three 48-hour periods. The day of blastocyst formation affected how much IFN-tau was produced during the first two culture periods, but not during the third period. The overall secretion of IFN-tau during the 6-day period increased significantly and well beyond what could be accounted for by the concomitant increase in cell numbers. In the second experiment, blastocysts were initially cultured in individual droplets for 48 hr, then plated into 48-well plates. Medium concentrations of IFN-tau were determined after 48 hr and again after 6 and 12 days of culture. Initial IFN-tau secretion did not affect the ability to form outgrowths or their final size, and initial differences in secretion between groups of blastocysts had disappeared by the second and third analyses. In the third experiment, blastocysts were cultured individually for 48 hr in droplets containing the medium that had been flushed through the uteri of non-pregnant sheep on days 10, 12, and 15 of the estrous cycle. Culture in the medium obtained from the Day 15 flush significantly increased the number of cells that blastocysts contained, as well as IFN-tau secretion.

Single daily intramuscular injections of low quantities of recombinant ovine interferon-tau extends luteal life-span in Angora goats.

The objective of this study was to determine whether single, daily intramuscular injections of low amounts of ovine interferon-tau (ovIFN-tau) would extend luteal life-span in nonpregnant Angora goats. Female goats were assigned randomly to receive a single daily injection of 1) PBS (control; n = 11), 2) 125 microg/d ovIFN-tau (n = 11), or 3) 500 microg/d ovIFN-tau (n = 11) from d 14 to 20 after estrus. Luteal life-span was defined as the number of days from the synchronized estrus until serum progesterone (P4) declined (< 0.5 ng/mL) and was of normal duration in controls (19.4 +/- 0.3 d) but was increased (P < 0.05) in goats receiving 125 microg/d (23.2 +/- 1.3 d) and 500 microg/d (25.5 +/- 1.2 d) ovIFN-tau. Injection of either ovIFN-tau dose caused an initial decrease (P < 0.05) in serum P4 concentrations relative to controls but did not differ from controls thereafter. Rectal temperatures increased (P < 0.05) following ovIFN-tau treatment until d 18 for goats given the lower dose and throughout the treatment period for those given 500 microg/d. In summary, injections of as little as 125 microg/d of ovIFN-tau extended luteal life-span in goats. This dose caused a transient reduction in serum P4 concentrations and induced hyperthermia.

Caprine pregnancy-associated glycoproteins (PAG): their cloning, expression, and evolutionary relationship to other PAG.

Pregnancy-associated glycoproteins (PAG) are structurally related to aspartic proteinases and belong to an extensive, rapidly evolving family of recently duplicated genes expressed in the placentas of artiodactyl species. The aim of the present study was to clone PAG from the goat, study their temporal and cell-specific expression, and determine their phylogenetic relationship to PAG from other species. RT-PCR was used to generate PAG cDNA from pooled placental RNA obtained between days 45 and 115 of pregnancy. A total of 11 cDNA, which differed by > 5% from each other, were selected for complete bidirectional sequencing from 60 clones analyzed. A group of nine (caPAG1, caPAG3-7(var), caPAG9-11), which displayed > 80% sequence identity with each other, were expressed after day 45 of pregnancy and were localized to trophoblast binucleate cells. These PAG demonstrated an unusually high ratio of nonsynonymous (amino acid changing) to synonymous nucleotide differences. CaPAG2, by contrast, was detectable only in early pregnancy (days 18 and 19) and expressed throughout trophectoderm. It was of more ancient origin than the PAG1 group, but more recent than caPAG8. The latter was expressed at all stages examined (days 18 to 115). The data confirm that many PAG genes, with different patterns of temporal and spatial expression, are transcribed in the placenta of the goat. The data also suggest that the recently duplicated PAG genes are being selected for rapid diversification of function.

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).

Conception rates in early postpartum ewes bred naturally or by intrauterine insemination.

Ewes were treated with a medroxyprogesterone acetate (MAP) sponge for 8 d followed, at sponge removal, with 500 IU pregnant mare serum gonadotropin (PMSG) at d 30, 40 or 50 (d 0 = lambing) to induce estrus. Dry and lactating ewes were divided into equal numbers at each postpartum day and bred at estrus. Conception rates and number of accessory sperm were determined by flushing the oviducts 3 d after mating and examining the recovered ova. There was no effect (P greater than .05) of lactational status on conception rates. Conception rates increased (P less than .05) from d 30 (10%) to d 40 (45%) and from d 40 to d 50 (80%). There were fewer (P less than .05) ova with accessory sperm (5/26) in d-30 ewes compared with d-40 (10/27) or d-50 (12/24) ewes. In Exp. 2, ewes were assigned to two groups after receiving PMSG on d 30: 1) mated naturally or 2) inseminated during laparotomy near the uterotubal junction (UTJ). Dry and lactating ewes were divided evenly within each of the two treatments. Oviducts were flushed and ova were examined for cleavage. The conception rate was 60% in ewes that were inseminated in the UTJ vs 10% in ewes mated to rams (P less than .05). Lactational status had no effect on results. In conclusion, conception rates in postpartum ewes treated with MAP sponge and PMSG increased from postpartum d 30 to d 50 with natural breeding, and d-30 conception rates were increased over natural mating by insemination into the uterine horn near the UTJ.

The effect of clenbuterol on time of lambing and neonatal death loss.

Two experiments were conducted to test the ability of clenbuterol, a beta-sympathomimetic agonist, to delay lambing. Ewes in all experiments were induced to lamb with 2.5 mg of flumethasone given intramuscularly on Day 141, 142 or 143 of gestation. Beginning on the day of flumethasone administration and continuing daily until lambing, ewes in Experment 1 were given injections intramuscularly (i.m.) with either 1) vehicle solution at 2200 h, 2) 240 ug of clenbuterol at 2200 h or 3) 240 ug of clenbuterol at 1600 and at 2200 h. There was a greater proportion of ewes lambing between 0800 h and 1600 h (P<0.01) and more stillbirths (P=0.05) in the groups which received clenbuterol than in the vehicle control group. Experiment 2 was conducted to determine if a lower dosage of clenbuterol, given as a single injection 37 h after flumethasone, would delay lambing without increasing the proportion of stillbirths. Ewes received either the vehicle solution, or 36 or 146 ug of clenbuterol. Lambing was delayed in the group which received 146 ug but not 36 ug of clenbuterol. There were no adverse effects of treatment on lamb survival in Experiment 2.

Effects of adrenalectomy and glucocorticoids on puberty in gilts reared in confinement.

The effect of adrenal function and flumethasone (FM, a synthetic glucocorticoid) on induction of puberty in crossbred gilts raised in confinement was examined in two experiments. In Exp. 1, gilts were adrenalectomized (Adx) or subjected to sham adrenalectomy (Sham) between 140 and 160 d of age. Twenty days later indwelling jugular catheters were implanted in Adx, Sham and another group of intact gilts designated as Controls, and the gilts were moved from confinement to outdoor pens and checked daily for estrus with a mature boar. Fewer (P less than .05) Adx (1/11) than Sham (9/14) gilts showed estrus and ovulated by 205 d of age. Response of Control gilts (6/14) was not different from the other groups. Although Adx gilts received 40 mg cortisone acetate and 10 mg deoxycorticosterone acetate daily throughout the experiment, mean plasma glucocorticoids were lower (P less than .05) in Adx (24 +/- 4.7 ng/ml) than in either Sham (47 +/- 8.1 ng/ml) or Control (44 +/- 6.1 ng/ml) gilts. Experiment 2 was conducted to determine whether FM given to Adx gilts immediately after surgery could have inhibited estrus and ovulation. Intact gilts received a total of 27.5 (FM1) or 17.5 (FM2) mg FM over 4 d between 150 and 160 d of age before relocation and boar exposure 20 d later. Control gilts received no injections. Nine of 13 FM-treated but none of the Control gilts showed estrus. It is concluded from these results that the adrenal glands may facilitate the onset of puberty in gilts through increases in glucocorticoid production, but that this is not required for puberty to occur.

Chromosomal evaluation of a ewe lamb with atresia ani vaginalis.

Chromosomes of a ewe lamb born with atresia ani vaginalis were examined after a 72-hour culture of peripheral whole blood. The 2n number of chromosomes was 54,XX, with no apparent deviation from normality. Pedigree analysis of the ewe lamb indicated that her sire and dam were only slightly related (Rsd = 0.59%); therefore, the amount of inbreeding of the lamb was small (Fx = 0.30%).

Lifespan of corpora lutea induced in estrous-synchronized cycling and anestrous ewes.

The effect of pretreatment with flurogestone acetate (FA) on the lifespan of corpora lutea induced with pregnant mare serum gonadotropin (PMS) was examined in cycling and anestrous ewes. Cycling ewes received one of three treatments: 750 IU PMS 2 d before expected estrus (P), FA-impregnated vaginal sponges for 16 d (F), and FA sponges for 16 d and 750 IU PMS 2 d before sponge removal (FP). A fourth group served as controls (C). When compared with d 12 means within treatment, plasma progesterone means were lower (P less than .05) on d 16 in control ewes, on d 15 in P and F ewes, and on d 14 in FP ewes. Only 44% of ewes receiving FA treatment alone exhibited estrus (P less than .05) compared with 100% of untreated ewes. The FP treatment increased ovulation rate compared with controls (P less than .01). The decrease in luteal lifespan observed in cycling ewes suggests a possibility of asynchrony between the uterus and embryo, which could result in failure of an embryo to prevent luteal regression, thus resulting in reduced fertility. None of the seasonally anestrous ewes that received PMS alone and only 55% of those treated with FA sponges for 8 d before PMS injection exhibited estrus. Ewes pretreated with FA exhibited higher plasma progesterone concentrations on d 10 through 16 after PMS injection. There were no differences in luteal lifespan as measured by peripheral plasma progesterone patterns. Although FA treatment did not alter luteal lifespan in anestrous ewes, the increased plasma progesterone concentrations observed with FA treatment suggest that progestogen pretreatment may be essential for optimal luteal function.