Follicular development,oocyte viability and recovery in relation to follicular steroids,prolactin and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge,no detectable LH surge,and progestin inhibition of LH surge

Estrous cycles of heifers (n = 137) were synchronized with prostaglandin (PGF_2α) and follicular development stimulated with follicle stimulating hormone. Twenty-eight animals were administered Norgestomet implants 12 hr prior to the initial PGF2α injection to suppress the LH surge that initiates ovulation. Animals were ovariectomized every 12 hr after the initial PGF2α (7–9/time, 12–108 hr and at 192 and 240 hr post PGF2α) and divided into three treatment groups to consist of: 1) animals exhibiting a normal luteinizing hormone (LH) surge (n = 86), 2) animals in which no LH surge was detected (n = 23), and 3) suppression of the LH surge via Norgestomet implants (72–108 hr, n = 28). Follicular diameter was measured and follicular fluid was collected for analysis of prolactin, estradiol, progesterone and glycosaminoglycan concentrations. Progesterone concentrations were increased in animals exhibiting an LH surge as compared to animals in which no LH surge was detected; primarily in large follicles (> 8 mm diameter) after the LH surge. Animals not exhibiting an LH surge also had increased follicular progesterone concentrations compared to Norgestomet-implanted animals (242.3 ± 36.3 vs 86.7 ± 6.4 ng/ml, respectively, P < .01), indicating some LH stimulation. Follicular estradiol in animals exhibiting an LH surge increased up to the time of LH surge detection and then declined whereas animals with no LH surge detected had follicular estradiol concentrations that declined after the PGF_2α injection. No differences were noted between those that did not exhibit an LH surge or in which the LH surge was suppressed with Norgestomet in relation to follicular estradiol concentrations. Follicular estradiol concentrations increased with follicular size in all treatment groups (P < .01). Follicular concentrations of prolactin were increased in small follicles (P < .05; ≤ 4 mm diameter) and follicular prolactin increased from 12 to 36 hr post PGF2α injection, then declined after the LH surge. Follicular glycosaminoglycan concentrations decreased with increases in follicular size (P < .01) and were higher in animals that did not exhibit an LH surge (P < .01). No differences in follicular glycosaminoglycans were noted between Norgestomet-implanted animals and those not exhibiting an LH surge. In the animals representing days 4 and 6 of the subsequent estrous cycle (192 and 240 hr post PGF2α), numbers of small-sized follicles were increased. Follicular progesterone and estradiol concentrations were related to atretic large follicles unovulated from the prior estrus and a wave of growth in small and medium follicles. Follicular prolactin and glycosaminoglycans increased with time of the new estrous cycle and were increased in smaller follicles (P < .01). Suppression of LH with progestin implants (Norgestomet) may relate to early effects of progesterone, which may not be totally eliminated at target tissues and subsequently alters the LH surge, steroidogenesis of the follicle, and ovulation. Oocytes were predominantly found in the follicular fluid from animals in which an LH surge was detected and in the buffer wash of follicles in which no LH surge was detected. Oocyte viability was higher in animals exhibiting an LH surge (75% viable) whereas the oocytes of Norgestomet-implanted animals were 75% degenerate.     

Effects of cytokines on FSH-induced estradiol production by bovine granulosa cells in vitro: Dependence on size of follicle

The objective of the present studies was to determine the effect of cytokines on FSH-induced estrogen production by granulosa cells from small (1–5 mm) and large (≥ 8 mm) bovine follicles. FSH-induced estradiol production by granulosa cells from large follicles (expressed as pg estradiol/10⁵ cells/24 hr) was not affected (P>.05) by 10 or 100 ng/ml of interleukin (IL)-1β, 10 or 100 ng/ml of tumor necrosis factor-α (TNFα) or 100 ng/ml of IL-2. In contrast, 100 ng/ml of IL-1β, IL-2 or TNFα inhibited (P<.05) FSH-induced estradiol production by 31%, 55% or 72%, respectively in cells from small follicles. Interferon-α (IFNα; 100 U/ml) inhibited (P<.05) FSH-induced estradiol production by 61% and 20% in cultures of cells from small and large follicles, respectively. Interferon-β (IFNβ; 100 U/ml), interferon γ (IFNγ; 100 U/ml) and bovine trophoblast protein-1 (bTP-1; 100 U/ml) inhibited (P<.05) estradiol production by 47%, 71% and 28%, respectively in cells from small follicles, but had no effect (P>.05) on FSH-induced estradiol production in cells from large follicles. TNFα binding protein-I blocked (P<.05) the inhibitory effect of TNFα on FSH-induced estradiol production by cells from small follicles. Viability of granulosa cells was not affected (P>.05) by the various cytokines. In summary, cytokines have little or no effect on FSH-induced estradiol production by bovine granulosa cells collected from large follicles, whereas cytokines (bTP-1 ≤ IL-1β < IL-2 = IFNβ < IFNα < IFNγ = TNFα) have potent inhibitory effects on FSH-induced estradiol production by granulosa cells collected from small follicles. Thus, it appears that less differentiated granulosa cells (small follicles) are more responsive to cytokines than are highly differentiated granulosa cells (large follicles).     

Estrogenic effects of genistein on reproductive tissues of ovariectomized gilts

The soybean phytoestrogen genistein has a range of estrogenic actions demonstrated in various species; however, only limited research has been done to investigate its effects in swine. The objective of this study was to characterize the effects of a graded dose of genistein on estrogen-sensitive uterine and cervical tissues in ovariectomized gilts. Thirty-four postpubertal gilts were ovariectomized and assigned randomly to 1 of 6 treatment groups 15 d postovariectomy. Treatment groups received vehicle, estradiol benzoate (2 mg/d), or genistein (50, 100, 200, or 400 mg/d) via intramuscular injection at 12-h intervals for 10 d. Following the treatment period, gilts were euthanized, and uterine and cervical tissues were collected and processed for chemical or histological analysis. Uterine and cervical tissue mass, as indicated by wet, dry, and protein weights and total DNA content (expressed per 100 kg of BW), increased as the dosage of genistein increased (P < 0.001 for each regression). Uterine and cervical wet weights were increased by a dosage of 200 mg of genistein/d (P < 0.001 and P < 0.01, respectively) but not by 100 mg of genistein/d (P = 0.38 and P = 0.14, respectively) compared with those of control gilts. Height of epithelial cells lining the uterine glands and the lumen of uterus and cervix increased when gilts were treated with estradiol benzoate or 400 mg of genistein/d (P < 0.01). When the gilts were treated with estradiol benzoate or 400 mg of genistein/d, immunohistochemical staining demonstrated an increase in the percentage of cells that stained positive for progesterone receptor in the uterine glands and in the cells lining the vaginal cervix (P < 0.05). In gilts treated with 400 mg of genistein/d, the percentage of cells stained positive for proliferating cell nuclear antigen increased in the epithelium of the uterine glands, uterine lumen, and vaginal cervix (P < 0.05). Tissue growth was stimulated by genistein in a dosage-dependent manner, although no dosage of genistein induced a response as great as that of estradiol benzoate. Estrogen-sensitive tissues of the ovariectomized gilt, such as the cervix and uterus, are affected by injection of large dosages of the phytoestrogen genistein. The sensitivity of the uterus of the gilt to estrogenic substances makes it a potential model to examine the impact of environmental endocrine modulators on reproductive tissues.     

Plasma bovine placental lactogen concentration throughout pregnancy in the cow; relationship to stage of pregnancy,fetal mass,number and postpartum milk yield

This study characterized the peripheral plasma placental lactogen (bPL) profile throughout gestation and examined the relationship between the stage of gestation, fetal mass, number, and postpartum lactation with circulating levels of bPL in Holstein cows after nonsurgical embryo transfer. Cows (n = 12) were divided into two groups: Group 1 = single embryo recipient cows (n = 5); Group 2 = twin embryo recipient cows (n = 7). Blood was collected about every third day from Day 0 (Day 0 was defined as the first day of standing estrus), then daily for the last 10 d of gestation, and sampling was stopped 1 d postpartum. The cows were milked twice daily at 0800 and 1800 hr. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from that of the group and reported separately. The time trend concentrations of plasma bPL were significantly affected by the stage of gestation (P < 0.01) but not fetal number (P < 0.21). In both groups bPL levels remained low during the first two trimesters, then increased rapidly (P < 0.01) to peak concentrations between Days 200 and 220, and stabilized at this elevated level until parturition. Postpartum milk yields were indistinguishable between the singleton and twin-bearing cows. Calf birth weight and postpartum lactation were both correlated (P < 0.01) to peripheral bPL concentration in singleton cows, however, this relationship decreased with a subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited a deviating bPL profile. These results indicate that peripheral bPL levels are positively associated with the stage of gestation but not with fetal number. Otherwise, the peripheral pattern of bPL is a valuable index for predicting feto-placental viability.     

Effect of somatotropin on adipose tissue metabolism: Ontogeny of the enhanced response to adrenergic challenge in the lactating cow

Seven multiparous Holstein cows (>150 d postpartum) were used to evaluate the time course of the chronic adaptation in lipolytic response to adrenergic challenge with bovine somatotropin (bST) treatment. Cows received daily bST (sometribove; 40 mg/d) or excipient injections for 7 d (single reversal design) with a 7-d interim between periods. Epinephrine challenges (1.4 μg/kg body weight intravenously) were administered on Days −2, −1, 1, 2, 3, 5, and 7 of treatment at 10:00 a.m. (15 hr after bST or excipient injection). Frequent blood samples were collected, and concentrations of plasma glycerol (GLY) and nonesterified fatty acids (NEFA) were determined. Treatment with bST increased milk yield 23% (P < 0.05) and milk fat content 33% (P < 0.001) compared with controls. Somatotropin-treated cows entered negative energy balance by Day 3 and had higher basal plasma concentrations of GLY and NEFA than did controls by Day 2 and Day 3, respectively. Response to epinephrine, expressed as area under the response curve corrected for basal, was enhanced by bST treatment, regardless of energy balance. GLY response was greater than control by Day 1 of bST treatment (P < 0.01), and had plateaued by Day 2 (P < 0.001). The NEFA response area was higher than control and had plateaued by Day 1 of bST treatment (P < 0.001). Day 1 represented 15 hr after the first bST injection. Results illustrate that bST treatment results in enhanced in vivo lipolytic response to catecholamine challenge, and the metabolic adaptation is in place by 15 hr after the first bST injection.     

The effect of pulsatile gonadotropin-releasing hormone and estradiol administration on luteinizing hormone and follicle-stimulating hormone concentrations in pituitary stalk-sectioned ovariectomized pony mares

Hourly pulses of gonadotropin-releasing hormone (GnRH) or bi-daily injections of estradiol (E2) can increase luteinizing hormone (LH) secretion in ovariectomized, anestrous pony mares. However, the site (pituitary versus hypothalamus) of positive feedback of estradiol on gonadotropin secretion has not been described in mares. Thus, one of our objectives involved investigating the feedback of estradiol on the pituitary. The second objective consisted of determining if hourly pulses of GnRH could re-establish physiological LH and FSH concentrations after pituitary stalk-section (PSS), and the third objective was to describe the declining time trends of LH and FSH secretion after PSS. During summer months, ovariectomized pony mares were divided into three groups: Group 1 (control, n = 2), Group 2 (pulsatile GnRH (25 μg/hr), n = 3), and Group 3 (estradiol (5 mg/12 hr), n = 3). All mares were stalk-sectioned and treatment begun immediately after stalk-section. Blood samples were collected every 30 min for 8 h on the day before surgery (DO) and 5 d post surgery (D5) to facilitate the comparison of gonadotropin levels before and after pituitary stalk-section. Additionally, jugular blood samples were collected every 12 hr beginning the evening of surgery, allowing for evaluation of the gonadotropin secretory time trends over the 10 d of treatment. On Day 10, animals were euthanized to confirm pituitary stalk-section and to submit tissue for messenger RNA analysis (parallel study). Plasma samples were assayed for LH and FSH by RIA. Mean LH secretion decreased from Day 0 to Day 5 in Groups 1 and 3, whereas LH secretion tended (P < 0.08) to decrease in Group 2 mares. On Day 5, LH was higher (P < 0.01) in Group 2 (17.26 ± 3.68 ng/ml; LSMEANS ± SEM), than either Group 1 (2.65 ± 4.64 ng/ml) or group 3 (4.28 ± 3.68 ng/ml). Group 1 did not differ from Group 3 on Day 5 (P < 0.40). Similarly, mean FSH levels decreased in all groups after surgery, yet Group 2 mares had significantly (P < 0.001) higher FSH concentrations (17.66 ± 1.53 ng/ml) than Group 1 or Group 3 (8.34 ± 1.84 and 7.69 ± 1. 63 ng/ml, respectively). Regression analysis of bi-daily LH and FSH levels indicated that the time trends were not parallel. These findings indicate: 1) Pituitary stalk-section lowered LH and FSH to undetectable levels within 5 d after surgery, 2) pulsatile administration of GnRH (25 μg/hr) maintained LH and FSH secretion, although concentrations tended to be lower than on Day 0, and 3) E2 did not stimulate LH or FSH secretion.     

Suppression of somatotroph function induced by growth hormone treatment in neonatal pigs

The effect of recombinant porcine growth hormone (pGH) treatment on pituitary function was evaluated in young pigs. Piglets received intraperitoneal recombinant pGH implants (0.5 mg/d sustained release) or vehicle implants beginning at 3 d of age. Ten piglets were sacrificed at 4 and 6 wk of age (five piglets/treatment group) for the collection of pituitary glands, blood, and liver tissue. Blood samples also were drawn at 3 and 12 d of age. Serum concentrations of GH, prolactin (PRL), thyroid-stimulating hormone (TSH), insulin-like growth factor-1 (IGF-1) and IGF-2 were evaluated. Levels of IGF-1 and IGF-2 mRNA were determined in liver samples. Treatment with GH increased circulating levels of GH and IGF-1 (P < 0.01), but not PRL, TSH, or IGF-2. Hepatic IGF-1, but not IGF-2, mRNA levels were increased by pGH (P < 0.001). Cultured pituitary cells from each animal were challenged with 0.1, 1, and 10 nM GH-releasing hormone (GHRH); 2 mM 8-Br-cAMP; or 100 nM phorbol myristate acetate. The release of GH from cultured pituitary cells was stimulated by all secretagogues (P < 0.001). The secretion of GH, but not PRL or TSH, in culture was inhibited by previous in vivo GH treatment (P < 0.001). Similarly, cellular GH, but not PRL or TSH, content was lower in the GH-implant group (P = 0.005). Cell cultures from 6-wk-old piglets secreted more GH, but not PRL or TSH, than cultures from 4-wk-old piglets (P < 0.05). Likewise, cellular GH, but not PRL or TSH, content was greatest in cultures from 6-wk-old animals (P = 0.002). Piglet growth was not affected by exogenous GH treatment (P = 0.67). These results demonstrate that exogenous pGH treatment selectively down-regulates somatotroph function in young pigs.     

Maternal treatment with somatotropin alters embryonic development and early postnatal growth of pigs

A possible management strategy to alter fetal development and enhance sow productivity and progeny performance was examined by maternal administration of porcine somatotropin during early gestation. Eighteen crossbred gilts were bred naturally to boars of similar genetics, and pregnancy was confirmed between Days 21 and 24 of gestation by ultrasound. All animals were allowed ad libitum consumption of a 16% CP gestation diet through Day 21 of gestation and 3.0 kg/d for the remainder of gestation. Gilts were injected twice daily with 0 (n = 10) or 15 μg/kg body weight (BW) (n = 10; total, 30 μg/kg BW per d) pituitary-derived porcine somatotropin (pST) during Days 28 to 40 of gestation. Data were collected postmortem during embryonic, neonatal, and market-weight phases. At 41 d of gestation, pST treatment increased embryonic survival (87.9 versus 77.0%; P < 0.05) and embryo crown rump lengths (77.96 versus 65.14 mm; P < 0.01), but embryo weight was not altered (10.15 and 9.03 g; P > 0.10). Pigs from pST-treated gilts had increased (P < 0.01) crown rump lengths at birth (31.5 versus 30.4 cm) and 21 d (50.9 versus 48.4 cm). However, no differences were observed in birth or 21-d weights as a result of pST treatment (P > 0.10). Neonatal carcasses of progeny (20 kg BW) from the pST-treated gilts had heavier semitendinosus muscles (76.1 versus 66.0 g; P < 0.10), larger longissimus muscle cross-sectional area (10.1 versus 8.2 cm²; P < 0.05), longer sides (51.2 versus 47.9 cm; P < 0.001), and decreased 10th rib backfat (6.67 versus 8.64 mm; P < 0.001) compared with those of controls. Carcasses of market-weight progeny (100 kg BW) from pST-treated gilts had larger longissimus muscle cross-sectional area (P < 0.10), heavier trimmed loins (P < 0.10), and longer carcass sides (P < 0.05). Data are supportive of a hypothesis that mechanisms during early embryonic development are sensitive to manipulation through selected management strategies of the sow and that modifications of this strategy may serve as a model for the examination of molecular and cellular events controlling early embryonic growth.     

Responsiveness to adipogenic agents in stromal-vascular cultures derived from lean and preobese pig fetuses: an ontogeny study.

Primary cultures of stromal-vascular (S-V) cells from adipose tissue were used to evaluate characteristics of preadipocytes from lean and preobese fetuses at several ages (50, 75, and 110 d). In insulin-supplemented (1 microM) cultures (serum free) there was a significant age x fetal genotype interaction (P less than .01) for glycerol-phosphate dehydrogenase specific activity (GPDH); GPDH activity was genotype-dependent at 110 d (preobese greater than lean). The responses of S-V cultures (preadipocyte development) to 2% pig serum and to insulin (serum free) were similar. Main effects of genotype and age were significant (P less than .05) for protein levels in pig serum and insulin-treated cultures. There was a significant genotype x age (P less than .05) interaction for GPDH activity and protein levels in cultures treated with dexamethasone + 3-isobutyl-1-methylxanthine (DEX-IBMX). Treatment with DEX-IBMX induced more preadipocyte development in cultures from preobese fetuses than in cultures from lean fetuses at 110 d (P less than .05). The responsiveness of S-V cultures to DEX-IBMX (enhanced development) increased considerably between 50 and 75 d regardless of fetal genotype, but there was little response in cultures form 50-d fetuses. Preadipocyte development in lean and preobese fetuses diverged between 75 and 110 d, resulting in many more preadipocytes in preobese fetuses at 110 d. Therefore, S-V cells from preobese fetuses (late term) may be inherently more sensitive to adipogenic agents than S-V cells from lean fetuses.     

Effects of bisphenol A,diethylhexyl phthalate and pentabrominated diphenyl ether 99 on steroid synthesis in cultured bovine luteal cells

Bisphenol A (BPA), diethylhexyl phthalate (DEHP) and pentabrominated diphenyl ether 99 (PBDE 99) are environmental toxicants belonging to the endocrine disrupting compounds (EDCs). They exert adverse effects on the various physiological systems, especially the reproductive system of humans and animals. The aim of this study was to investigate the effects of BPA, DEHP and PBDE 99 on progesterone (P4) synthesis in cultured bovine luteal cells. The bovine luteal cells isolated from the mid-luteal corpora lutea were exposed to different concentrations of BPA (1, 3, 10 and 30 µM), DEHP (1, 3, 10 and 30 µM) and PBDE 99 (0.1, 0.3, 1 and 3 µM) in a serum-free culture media for 48 and 96 hr. At 48 hr, the P4 level in the luteal cells decreased after treatment with all concentrations of BPA; 3, 10 and 30 µM of DEHP; and 3 µM of PBDE 99 compared to the control (p < .05). Treatment of cells with 3–30 µM of BPA, 1–30 µM of DEHP and 1–3 µM of PBDE 99 for 96 hr resulted in reduction in P4 synthesis (p < .05). However, lower concentrations of PBDE 99 (0.1 and 0.3 µM) increased P4 levels at 48 and 96 hr. Synthesis of P4 was lower at 96 hr compared to the 48 hr in the groups treated with BPA (30 µM), DEHP (1–30 µM), PBDE 99 (0.3–3 µM) and control group. Our results showed that BPA, DEHP and PBDE 99 are able to alter luteal steroidogenesis in bovine cells and can disrupt hormonal balance in the ovary. However, it is necessary to evaluate the exact mechanism underlying these effects in future studies.     

Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin‐based diluent

Antioxidants are known to prevent the reactive oxygen species (ROS)‐mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid–protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll^®. Sperm pellet was resuspended in soya lecithin–Tris–fructose diluent (400 × 10⁶ cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3–5°C for 72 hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo‐osmotic swelling test (HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X‐100 were resolved by SDS‐PAGE and quantified using Quantity One software (Bio‐Rad, USA). The rapid motility, linearity and straight‐line velocity (VSL) were found significantly (p < .05) higher in the antioxidant‐treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant‐treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower (p < .05) in the sperm extract of antioxidant‐treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase (p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant‐treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage.     

Incidence and Prevention of Early Parturition in Sows

A retrospective study, based on 60 990 farrowing records from 35 commercial herds, was performed to determine the incidence of early parturition (<114d) and to investigate the relationship between early parturition and total number of piglets born, number of piglets born alive and percentage of stillborn piglets per litter. The mean gestation length was 115.4 ± 1.62d, and early parturition occurred in 10% of all farrowing records. Sows with early parturition had significantly more stillborn piglets and a larger litter size compared to sows with a normal gestation length (114–117d). Sows with a gestation length <114d were 1.2 times (95% CI: 1.19–1.21; p < 0.001) more likely to have an early parturition at the subsequent parity. A second study was performed in four herds (n = 329) to investigate the efficacy of altrenogest administration on 110–112d (T112) or 111–113d (T113) of gestation for preventing early parturition and to investigate the effect of this treatment on the reproductive performance of sows. The interval between the last altrenogest treatment and the onset of parturition was 3.3 ± 1.32 (T112) or 2.0 ± 0.89 (T113) days. The gestation length of sows of the altrenogest group (T112 + T113) (115.3 ± 1.23d) was significantly longer compared to gestation length of the non‐treated sows (114.7 ± 1.69d) (p < 0.01). Altrenogest treatment had no negative effect on the reproductive performances of the sows. In conclusion, the administration of altrenogest in late gestation is an effective and safe method to prevent early parturition and can counteract the reproductive losses because of premature farrowing, which may occur in a substantial part of the farrowing events.     

Growth and compositional changes of fetal tissues in pigs

Three hundred twenty fetuses were obtained from 33 pregnant gilts (Camborough-22, Pig Improvement Co.) to determine rates of nutrient deposition in fetal tissues and to estimate nutrient requirements for fetal growth. Pregnant gilts were fed an equal amount of a gestation diet (2.0 kg/d; as-fed basis), and were slaughtered at d 0, 45, 60, 75, 90, 102, or 110 of gestation (n = 3 to 6 per day). Fetuses were dissected into carcass and individual tissues (including gastrointestinal tract, liver, lung, heart, kidney, spleen [> or = d 75]), and partial placental collection was made for chemical analysis. Fetal tissues were weighed and analyzed for DM, ash, CP, and crude fat. Regression equations were obtained to explain the weight and compositional changes of individual tissues during gestation. Weights of the fetus, carcass, gastrointestinal tract, liver, heart, lung, and kidney increased cubically (P < 0.001), whereas brain weight increased linearly (P < 0.001) as gestation progressed. Fetal protein and fat contents increased quadratically (P < 0.001) as gestation progressed (R2 = 0.906 and 0.904, respectively). Changes in fetal protein and fat contents fit a multiphasic regression that consisted of two linear equations (P < 0.001, R2 = 0.988 and P < 0.001, R2 = 0.983, respectively), indicating that protein and fat growth accelerated after d 69 of gestation. Fetal protein and fat accretions were 0.25 and 0.06 g/d (P < 0.001) before d 69 of gestation, and increased to 4.63 and 1.09 g/d (P < 0.001) after d 69 of gestation. Protein needs for tissue protein gains increased 19-fold after d 69 of gestation. Results of this study indicate that the growth of the fetus and fetal tissues occurs at different rates during gestation and support the practice of a two-phase feeding strategy (before and after approximately d 70 of gestation) for pregnant gilts.     

Partial Purification and Characterization of Two Non-FSH Steroid-Modulating Factors in Rat Thymic Epithelial Cell-Conditioned Medium (TCM)

Previously, we have reported that unknown factor(s) in rat thymic epithelial cell-conditioned medium (TCM) stimulates basal and follicle-stimulating hormone (FSH)-induced steroid hormone production and aromatase enzyme activity in cultured rat granulosa cells. Here we report the partial purification and characterization of two of these activities. Thymic epithelial cells were prepared from immature female rats and used for TCM production. Lyophilized aliquots of TCM were reconstituted with distilled water at 25% of the original volume, applied to a gel filtration column, and column fractions were tested for their stimulation of steroidogenesis in granulosa cells prepared from immature diethylstilbestrol-treated rats. Two distinct biologically active regions were identified that corresponded to apparent molecular weights of approximately 22,000 and less than 1,000. The <1 kDa activity (“TCM-1”) stimulated (P < 0.01) basal production of progestins [progesterone and 20α-hydroxypregn-4-en-4-one (20α-OH-progesterone)] and estrogen, and also induced dramatic morphological changes on the rat granulosa cells. In contrast, the ∼22 kDa activity (“TCM-22”) stimulated (P < 0.01) only basal progestins, and had no effect (P < 0.05) on basal estrogen production or morphology of the cultured rat granulosa cells. In the presence of 100 ng/ml FSH, TCM-1 stimulated (P < 0.01) estradiol and progesterone production, whereas TCM-22 stimulated (P < 0.01) progesterone, but inhibited (P < 0.01) estradiol production. When both activities were assayed together, they were synergistic in stimulating (P < 0.01) basal progesterone production, but TCM-22 antagonized (P < 0.01) TCM-1-induced estradiol production. The biologic and physico-chemical characteristics of TCM-1 and TCM-22 were distinct from one another, as well as from FSH. When subjected to C₈ reverse-phase HPLC, TCM-1 retained its characteristic biologic properties and was eluted (54% acetonitrile) as A₂₁₄-absorbing moiety with a peak retention time of 92–93 minutes. The elution of TCM-22 was not correlated with an identifiable protein peak. These results suggest that ovarian steroid production may be modified by non-FSH factors produced by thymic epithelial cells although amino acid sequencing of TCM-1 was unsuccessful. This highlights a potential role of the thymus gland in regulating ovarian function.     

Vaccination of Rainbow Trout against Infectious Hematopoietic Necrosis (IHN) by Using Attenuated Mutants Selected by Neutralizing Monoclonal Antibodies

Abstract

A neutralizing monoclonal antibody against infectious hematopoietic necrosis virus (IHNV) was used to select neutralization-resistant mutants from isolates of virus obtained from adult steelhead Oncorhynchus mykiss returning to the Round Butte Hatchery (RB mutants) on the Deschutes River in Oregon, USA, and from rainbow trout (nonanadromous O. mykiss) at a commercial hatchery in the Hagerman Valley of Idaho, USA (193-110 mutants). Two of the mutants, RB-1 and 193-110-4, were significantly (P < 0.001) attenuated compared with parental strains. Vaccination of rainbow trout by waterborne exposure to the mutants conferred solid protection against challenge with wild-type virus. In some trials, fish vaccinated with the RB-1 mutant at 50% tissue culture infectious doses (TCID50) of 1 × 10⁴–1 × 10⁵ TCID50/mL or with the 193-110-4 mutant at 1 × 10²–1 × 10³ TCID50/mL, held for 14 d, then challenged with the homologous wild-type strain at 1 × 10⁵ TCID50/mL showed relative percent survival of 95–100% (P < 0.005). There was no significant difference (P > 0.05) in protection among fish exposed to the RB-1 vaccine strain at a dose of 1 × 10⁵ TCID50/mL for periods of either 1, 12, or 24 h, held for 14 d, and then challenged with the wild-type RB isolate, although the 1-h exposure seemed to be somewhat less effective. Fish were vaccinated with the RB-1 strain at 1 × 10³–1 × 10⁵ TCID50/mL for 24 h then challenged after 1, 7, 14, or 21 d with the wild-type RB isolate. No significant (P > 0.1) protection was observed at 1 d postvaccination, but the relative percent survival increased progressively at each subsequent challenge period, becoming statistically significant by day 7 (P < 0.001) and beyond. These results suggested that resistance to challenge with wild-type virus resulted from development of IHNV-specific immunity and not from viral interference or interferon induction, and they reinforce the potential of an attenuated vaccine to control this important disease.     

Effect of Early Postpartum Ovulation on Fertility in Dairy Cows

Our objectives were to determine the effects of early ovulation on fertility and uterine health of dairy cows. Four hundred and forty‐five Holstein cows (185 primiparous and 260 multiparous) from five herds were used. Blood samples were collected at 21, 35 and 49 days in milk (DIM) and cows were considered to be cyclic at 21 DIM (Cyc21) if serum progesterone (P4) concentration was above 1 ng/ml, cyclic by 49 DIM (Cyc49) if P4 concentration was above 1 ng/ml at 35 or 49 DIM, or not cyclic (NotCyc) if P4 concentration was below 1 ng/ml at all sample times. Endometrial cytology for diagnosis of subclinical endometritis was examined at 49 DIM in a subset of 414 cows. Cows in the group Cyc21 had increased hazard of insemination, for the first service, compared with cows in Cyc49 [hazard ratio (HR) = 1.40; 95% CI = 1.10–1.79; p = 0.006] and NotCyc (HR = 2.07; 95% CI = 1.52–2.82; p < 0.001). Cows in the Cyc49 group also had increased hazard of insemination compared with cows in the NotCyc group (HR = 1.47; 95% CI = 1.13–1.93; p = 0.005). Median days to insemination were, respectively, 71, 76 and 96 for cows in Cyc21, Cyc49 and NotCyc groups. Cows in Cyc21 had greater first service pregnancy per AI than Cyc49 [38.6 vs 28.1%; adjusted odds ratio (AOR) = 1.67; 95% CI = 1.01–2.75; p = 0.04] and NotCyc (38.6 vs 23.6%; AOR = 2.08; 95% CI = 1.08–4.00; p = 0.03). Pregnancy per AI was similar in Cyc49 and NotCyc cows (28.1 vs 23.6%; AOR = 1.25; 95% CI = 0.70–2.24; p = 0.45). Cows in Cyc21 had increased hazard of pregnancy up to 300 DIM compared with Cyc49 (HR = 1.52; 95% CI = 1.17–1.96; p = 0.002) and NotCyc (HR = 1.98; 95% CI = 1.41–2.78; p < 0.001). Cows in Cyc49 tended to have increased hazard of pregnancy compared with NotCyc (HR = 1.31; 95% CI = 0.96–1.77; p = 0.09). Median days to pregnancy were, respectively, 103, 147 and 173 for cows in Cyc21, Cyc49 and NotCyc groups. Cows in the Cyc21 group had decreased prevalence of subclinical endometritis compared with cows in the NotCyc group (29.9 vs 43.7%; AOR = 0.53; 95% CI = 0.29–0.97; p = 0.04); however, the prevalence did not differ from the Cyc49 group (29.9 vs 39.1%; AOR = 0.68; 95% CI = 0.41–1.14; p = 0.15). Cyc49 cows had similar prevalence of subclinical endometritis compared with NotCyc cows (AOR = 0.77; 95% CI = 0.46–1.29; p = 0.32). Early postpartum ovulation was associated with improved uterine health and fertility.     

Resynchronization of ovulation with new and reused intravaginal progesterone‐releasing devices without previous pregnancy diagnosis in Bos taurus indicus cows subjected to timed‐artificial insemination

The study aimed to evaluate pregnancy per artificial insemination (P/AI) of cows subjected to synchronization and resynchronization in ovulation protocols using intravaginal progesterone‐releasing insert (P4) before pregnancy diagnosis (PD) and the relationship of PR with the diameter of preovulatory follicles (ØPOF) before TAI. Cows (n = 378) were distributed into two groups: a resynchronization group with new devices (GRN; n = 185) and resynchronization group with used devices (GRU; n = 193). On Day 0, both groups received a new P4 and estradiol benzoate (EB). On D8, P4 removal + D‐cloprostenol + eCG + estradiol cypionate (EC) was done. On d10, TAI was conducted. On d32, cows were resynchronized and divided into two groups, GRN (n = 185) and GRU (n = 193). The GRN group received a new P4 + EB, and the GRU group received a used P4 + EB. On d40, the P4 was removed + PD. The non‐pregnant cows received D‐cloprostenol + eCG + EC. US was done again on d42 to determine ØPOF before the second TAI. The P/AI of the GRN and GRU groups after synchronization were 56.2% and 57.0% (p = 0.87), respectively, and those after resynchronization were 58.0% and 37.3% (p < 0.008), respectively. The P/AI of the GRN and GRU groups observed after TAI (synchronization + resynchronization) were 81.6% and 73.1%, respectively (p = 0.047). No difference (p = 0.067) in ØPOF between the pregnant and non‐pregnant cows in the GRN was found, whereas the GRU group showed a significant difference (p = 0.003). Resynchronization protocols optimized the P/AI in both groups. New intravaginal devices resulted in greater P/AI and P/AI accumulation in resynchronization as compared with the GRU; the ØPOF was related with P/AI.     

Cumulus and oocyte maturation and in vitro and in vivo fertilization of oocytes in relation to follicular steroids,prolactin, and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge,no detectable LH surge,and progestin inhibition of LH surge

Crossbred heifers (n = 103) were synchronized to estrus with prostaglandin (PGF_2α) and superovulated with follicle stimulating hormone (FSH-P). Animals were ovariectomized every 12 hr after the PGF_2α injection (n = 7 to 9/time) up to 108 hr to monitor the follicular, hormonal, and oocyte changes associated with follicular development and ovulation. Twenty-eight animals were implanted with Norgestomet implants 12 hr before PGF_2α and ovariectomized at 72, 84, 96, and 108 hr post PGF_2α injection to monitor effects of progesterone and suppression of the luteinizing hormone (LH) surge on oocyte maturation and quality. Follicular fluid was collected and analyzed for progesterone, estradiol, prolactin, and glycosaminoglycan content in conjunction with cumulus maturation and nuclear stage of oocyte maturation. Analysis of in vivo matured oocytes by in vitro fertilization was carried out at 60, 72, 84, and 96 hr post PGF_2α and in vitro matured oocytes at 12 to 108 hr post PGF_2α. No developmental changes in cumulus cells surrounding the oocyte of small follicles was noted (≤ 4 mm dia) indicating a static population. Medium (> 4 ≤ 8 mm) and large size (> 8 mm) follicles developed to the corona radiata and loose cumulus stages in animals in which an LH surge was detected but cumulus status remained primarily in the tight cumulus stage for animals without an LH surge. The estradiol-to-progesterone ratio for tight cumulus (TC), corona radiata (CR), and loose cumulus (LC) stages was 1.8 ± .1, 1.0 ± .1, and .4 ± .2, respectively (P < .01). Nuclear maturation of oocytes in small follicles from animals without a detectable LH surge seem to indicate early maturation (48 to 72 hr post PGF_2α) in conjunction with a high percent of degenerate oocytes not seen in animals exhibiting an LH surge. Oocytes from medium size follicles matured to germinal vesicle breakdown (GVBD) and early meiosis (metaphase I; MI) stages of development in all treatments. Most oocytes were degenerate in Norgestomet-implanted animals. Oocytes from large follicles (> 8 mm dia) from animals exhibiting an LH surge were in MI and metaphase II (MII) stages (48 to 84 hr post PGF_2α) in preparation of ovulation whereas oocytes from animals not exhibiting an LH surge had oocytes that early matured to MII (48 to 72 hr post PGF_2α), later regressing to degenerate oocytes (84 to 108 hr). Follicular progesterone, estradiol, and prolactin increased with oocyte maturation, particularly in medium and large follicles. In vivo matured oocytes for fertilization (60, 72, 84, and 96 hr post PGF_2α) were nude (from the oviduct) and primarily CR from follicles. Tubal oocytes (37%) were fertilized more frequently by a single sperm than follicular oocytes (14.3%; P < .01) and single sperm penetration peaked at 72 hr post PGF_2α. Follicular hormone concentrations were not related to sperm penetration. Oocytes (n = 101) matured in vivo had lower fertilization potential from ovaries producing < 14 or > 50 follicles (39.3%) as compared to 21 to 45 aspirated follicles (68.2%; P < .05), with a peak penetration at 32 follicles (86.7% penetration). No treatment differences (LH surge or no detectable LH surge) were noted in relation to in vivo matured oocytes. Oocytes with single sperm penetration had the lowest estradiol/progesterone ratio of 2.2 vs polyspermic penetration of 13.7.     

Effects of body condition and estradiol on luteinizing hormone secretion in post-partum beef cows

An experiment was conducted to test the hypothesis that the effect of body fatness on LH pulsatility in post-partum cows is entirely independent of the negative feedback effects of ovarian steroids. Forty beef cows were fed in the last 100 d of gestation so that they achieved either a thin (mean score 1.97) or fat (mean score 2.79) body condition (0 to 5 scale) at calving and were fed after calving to maintain live weight and body condition. At 15 (sd 3.7) d post partum all cows were ovariectomised and half from each body condition score treatment group received a subcutaneous estradiol implant (+EST) while the remainder received no implant (−EST). At weeks 5 and 9 post-partum blood samples were collected via jugular catheter every 20 minutes for 10 hr on two consecutive d and on the third d cows were injected via the jugular vein with 2.5 μg GnRH. Blood samples were collected every 15 minutes for 1 hr before and 2 hr after GnRH injection. At 5 and 9 weeks the fatter cows had significantly higher mean LH concentrations, baseline LH concentrations, LH pulse amplitudes and pulse frequencies (P<0.01). Implantation with estradiol in both fat and thin cows reduced mean LH concentrations, baseline LH concentrations, LH pulse amplitudes and pulse frequencies (P<0.001). The lack of interaction between body condition and the presence or absence of estradiol implies that the effect of body condition on LH release is independent of ovarian steroid feedback mechanisms. Fat cows showed a greater release of LH in response to exogenous GnRH (P<0.01) than thin cows while implantation with estradiol in both fat and thin cows decreased (P<0.01) LH release. The pituitary responsiveness to GnRH with the −EST cows was greater at 9 compared to 5 weeks, but there was no difference with time in the +EST cows. However, there was no such interaction in endogenous LH pulse amplitude suggesting that in the absence of estradiol the magnitude of GnRH pulses declined with time post-partum.     

Ghrelin antagonist D-Lys3-GHRP-6 counteract ghrelin effects in bovine cumulus-oocytes complexes matured in vitro

Ghrelin is a gut hormone related to energy balance and reproductive functions. The aim of this study was to evaluate the effect of ghrelin antagonist D-Lys3-GHRP-6 (GA) as a potential agent that prevents ghrelin effects during bovine oocyte maturation on progesterone production, cumulus cell (CC) viability, CC DNA damage and embryo development and hatching rates. Ghrelin's potential to induce oxidative stress in cumulus-oocyte complexes (COC) was also evaluated. COCs were cultured for 24 hr in medium without supplementation (C) or supplemented with 60 pM ghrelin (Ghrelin60), Ghrelin60 + 20 pM GA (GA20), Ghrelin60 + 60 pM GA (GA60) or Ghrelin60 + 100 pM GA (GA100) for experiment I. For experiment II, C and Ghrelin60 treatments were used. Differences between C and Ghrelin60 and the linear or quadratic association between GAs on Ghrelin60 were evaluated. Results demonstrated that Ghrelin60 increased progesterone concentration, reduced CC viability, induced CC DNA damage and decreased blastocyst and hatching rate compared with C (p < .05). GA20, GA60 and GA100 had a linear effect on CC genetic damage index (p ≤ .05) and a quadratic effect on CC viability (p < .01). GA20 counteracted the low hatching rate produced by Ghrelin60. However, GAs did not counteract progesterone concentration and blastocyst rate (p ≥ .21). GRH60 did not differ from C in the oxidative status (p ≥ .19). Our study highlights that GA could prevent the negative effects of ghrelin during bovine IVM.