Research Note: Development of a sandwich ELISA for determining plasma growth hormone concentrations in goose.

Growth hormone (GH) is required for normal postnatal development in poultry; however, no immunoassay exists to assess its levels in geese plasma, hindering the study of endocrine regulation in this species. We developed a sandwich ELISA to determine the GH concentrations in the plasma of geese. Recombinant goose GH was produced using a eukaryotic expression system and purified for use as the reference standard in ELISA and the antigen for producing the polyclonal antibodies in rabbits. Rabbit anti-goose GH polyclonal antibody was used to coat the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and catalyze the chromogenic reaction of 3,3,5,5-tetramethylbenzidine and H2O2. A sigmoidal curve was fitted to the optical density and the log of the standard GH concentration using the four-parameter logistic model. The sensitivity of the assay was less than 0.156 ng/mL. The intra- and interassay coefficients of variation were less than 9 and 13%, respectively. The response curve of the serially diluted plasma samples from geese exhibited a good parallel relationship with that observed for the reference standards. The assay effectively detected differences in GH concentrations in plasma samples from geese at various physiological stages; thus, it will be useful for future study of their growth and metabolism.

Improvement of goose embryonic and muscular developments by wider angle egg turning during incubation and the regulatory mechanisms.

Egg turning during incubation plays important roles in achieving high hatching performance and gosling quality. The objective of this study was to improve embryonic and muscular developments so to achieve better gosling quality by wider egg turning angles during incubation, and to unravel the associated regulatory molecular mechanisms. In each of three consecutive incubations, 1,728 goose eggs were divided into 3 groups that were set in the same type of commercial incubators with turning angles adjusted differently to 50°, 60°, and 70°, respectively. On average of the 3 tests, incubation with wider 70° turning angle reduced the post-18-day embryo mortality, promoted embryonic growth and development, improved the hatchability and gosling quality. On embryonic day of 29, gene mRNA expression levels of the hypothalamic growth hormone-releasing hormone (GHRH), pituitary growth hormone (GH), and liver insulin-like growth factor 1 (IGF-1) were higher in the 70° turning group than in the 50° or 60° groups. Wider angle turning also increased mRNA expression levels of the muscle development regulatory genes such as MYF5, MyoD, Myogenin (MyoG), and MRF4. Changes in expression of the above genes, together with the upregulation of the Pax3 and Pax7 genes in leg muscles, well explained the enhancement of the muscular growth and development when eggs were incubated by wider turning angles. These results also extended our understanding of the impacts and mechanisms of egg turning during incubation on hatching performance and gosling quality.

Protective effects of L-carnitine on reproductive capacity in rats with diabetes.

To explore the protective effects of L-carnitine on erectile function and reproductive function in rats with diabetes. A total of 60 male diabetes mellitus induced-erectile dysfunction (DMED) rats were randomly divided into three groups, 20 rats in each group. The blank group was fed normally, the control group was fed with 0.9% sodium chloride solution 5 ml/kg/day, and the experimental group was given L-carnitine 300 mg/kg/day. After six weeks, the Corpus cavernosum penis pressure (ICP) and mean arterial pressure (MAP) were measured. The sperm of epididymis were taken to detect the parameters of sperm. After six weeks of treatment, ICP and MAP in the experimental group were significantly higher than those in the control group and blank group (p < 0.05), and sperm density and PR in the experimental group were significantly higher than those in the control group and the blank group (p < 0.05). Superoxide dismutase (SOD) in the experimental group was significantly higher than that in the control group and blank group (p < 0.05). Malondialdehyde (MDA) in the experimental group was significantly lower than that in the control group and blank group (p < 0.05). The follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone in the experimental group were significantly higher than those in the control group and blank group (p < 0.05). We conclude that L-carnitine can significantly improve erectile function and reproductive function in rats with diabetes and it has great potential in the treatment of systemic organ damage in DMED rats.

Impairment of testes development in Yangzhou ganders by augmentation of leptin receptor signaling.

The aim of this study was to determine the cellular and molecular mechanisms of leptin (LEP) and the leptin receptor (LEPR) in testicular development of prepubertal ganders. In an in vivo animal experiment, active immunization against LEPR severely depressed prepubertal testicular development by significantly reducing testicular weights at 200 and 227 days of age. The number of elongated spermatids in the seminiferous tubules was also significantly decreased by immunization with LEPR at ages of 200 and 227 days. Inhibition of testicular development by LEPR immunization was associated with decreases in LHR, StAR, 3ß-HSD, CYP11A1, CYP17A1, and PRLR mRNA expression levels in testicular tissue, which resulted in a significant decrease in testosterone synthesis. In the in vitro experiments, the addition of LEP combined with anti-LEPR antibodies strengthened LEPR signal transduction, and inhibited significantly testosterone production in cultured Leydig cells isolated from prepubertal gander testes. The mRNA expression of LHR, StAR, 3ß-HSD, CYP11A1, CYP17A1 also decreased significantly after treatment with LEP combined with anti-LEPR antibodies in cultured Leydig cells. These results suggest that anti-LEPR antibodies strengthen LEPR signaling transduction in the presence of LEP, and immunization against LEPR inhibited testes development and testosterone secretion in prepubertal ganders.

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese.

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Cellular and molecular mechanisms of low dose prolactin potentiation of testicular development in cockerels.

The aim of this study was to determine the cellular and molecular mechanisms of prolactin (PRL) in testicular development of prepubertal cockerels. In an in vivo animal experiment, active immunization against PRL severely depressed prepubertal testicular development by significantly reducing testicular weights at both 122 and 164 d of age. The number of elongated spermatids in the seminiferous tubules was also significantly decreased by immunization with 199-residue chicken PRL (cPRL) at age 122 d. Inhibition of testicular development by cPRL immunization was associated with decreases in LH receptor (LHR), FSH receptor (FSHR), Stat5b, P450scc, steroidogenic acute regulatory (StAR) protein, and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression levels in testicular tissue. In in vitro experiments, testosterone production by cultured Leydig cells isolated from prepubertal cockerel testes was dose-dependently enhanced by treatment with bioactive recombinant PRL, but a lesser response was seen with high concentrations of PRL. The distinct changes in testosterone production in response to high and low concentrations of added PRL were paralleled by similar patterns of change in the mRNA levels of Stat5b, LHR, P450scc, StAR, 3ß-HSD, and CYP17A1 in cultured Leydig cells, as well as protein amounts of phosphorylated Jak2 and Stat5a/b. In conclusion, low to medium doses of PRL potentiate testis development in prepubertal cockerels by enhancing testosterone secretion from Leydig cells via activation of PRLR/Stat5b signal transduction, which upregulates mRNA expression of LHR and testosterone synthesizing enzymes. However, this positive regulation was weaker in response to a high dose of PRL, which reduced PRLR/Stat5b signal transduction and the expression of genes involved in LH signaling and testosterone synthesis.

Effect and molecular regulatory mechanism of monochromatic light colors on the egg-laying performance of Yangzhou geese.

Photoperiodic control is essential for manipulating the reproductive performance of avian species. This study was conducted to assess the neuroendocrine mechanisms that regulate reproductive functions of Yangzhou geese when there are different monochromatic light colors from light emitter diode (LED) sources. A flock of geese was divided into four groups with white, red, blue, and green light treatments being imposed. The results indicated that peak laying rates and reproductive performance were greater in geese treated with white or red as compared with blue or green light treatments. The fertilization rate of eggs and hatchability of fertilized eggs were greater with the white or red as compared with blue or green light treatments. There was a greater abundance of OPN5, Dio2, c-Fos, and GnRH-I mRNA in the hypothalamus earlier in the treatment period and abundances of these hypothalamic factors were greater with the white or red light treatments. Abundances of pituitary LH beta and FSH beta mRNA increased at a lesser rate with the blue or green light treatments and were in greater abundances with the white or red light treatments. The lighting regimen also resulted in photo-refractoriness with there being greater abundances of GnIH, VIP, and PRL mRNA with the use of white or red light treatments. The results indicate that the use of white or red monochromatic lights while imposing a long photoperiod of 11 h daily could result in sustaining functions of the reproductive system of Yangzhou geese for considerably longer times, thus, resulting in greater egg-laying performance.

Development of a sandwich ELISA for determining plasma prolactin concentration in domestic birds.

The present study was conducted to establish a sandwich ELISA for the determination of prolactin (PRL) concentrations in the plasma of domestic fowls. The assay uses a recombinant goose PRL as the reference standard, expressed in a eukaryotic system, and as the antigen for raising a polyclonal antibody in rabbit. This rabbit anti-goose PRL polyclonal antibody was used for coating the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and to catalyze the chromogenic reaction using 3,3',5,5'-tetramethylbenzidine as the substrate. The assay showed a linear relationship between the optical density and concentration of the standard PRL in the 0 to 12.5 ng/mL range, and the assay was sensitive to a concentration as low as 0.39 ng/mL. The intra- and inter-assay CVs were <7% and 11%, respectively. The response curves of the serially diluted plasma samples from goose, duck, and chicken exhibited similar parallel relationships to that observed for the reference standards. Consistent with previous findings, the assay effectively detected differences in PRL concentration in plasma samples from chicken, duck, and goose at various reproductive stages.

Endocrine and molecular regulation mechanisms of the reproductive system of Hungarian White geese investigated under two artificial photoperiodic programs.

Hungarian White geese are regarded as good producers of meat, eggs, and feathers, but specific lighting schedules are required to improve their egg-laying performance. This study reveals the neuroendocrine regulatory mechanisms that govern the reproductive activities and egg-laying performances of Hungarian White geese. The results indicated that increasing the daily photoperiod from a short 8 h period to either 11 h or 14 h initiated reproduction. Egg-laying rates increased faster in the 14 h group, peaking (48.2%) on day 33 as compared to the peak (52.67%) reached on day 53 in the 11 h group. Changes to the plasma estradiol and progesterone concentrations produced similar patterns in the two groups. In the hypothalamus, OPN5, Dio2, c-Fos, and GnRH-I expression levels showed similar sequential increases and decreases. Changes in GnIH and VIP expression levels were the opposite to those of GnRH-I, but the levels peaked earlier under the 14 h photoperiod conditions. Pituitary LH beta and FSH beta expression levels increased at slower rates but remained significantly higher in the 11 h group than in the 14 h group. However, pituitary PRL expression increased considerably earlier and was higher in 14 h geese than in 11 h geese, which was opposite to the observed egg-laying rate patterns. An increase from a short to a relatively long photoperiod (11 h) regulated the neuroendocrine system and led to reproductive activities being sustained for a longer period, which resulted in high egg-laying performances.

Anti-leptin receptor antibodies strengthen leptin biofunction in growing chickens.

Antibodies against the extracellular domains of the chicken leptin receptor were used to study the biological function of leptin in growing chickens. Both polyclonal and monoclonal anti-LEPR antibodies were administered intramuscularly to 30-d-old Chinese indigenous Gushi pullets. Both antibody preparations increased feed intake for 6 h after injection and reduced plasma concentrations of glucose, triglycerides, and both high- and low-density lipoproteins. The antibody treatments also upregulated agouti-related peptide and neuropeptide Y in the hypothalamus and downregulated proopiomelanocortin, melanocortin 4 receptor, and leptin receptor. The treatments also upregulated leptin receptor, acetyl CoA carboxylase beta, and acyl-CoA oxidase in the liver, abdominal fat, and breast muscle and downregulated sterol regulatory element-binding protein-1 and fatty acid synthase. Furthermore, even though the anti-leptin receptor antibodies failed to affect leptin receptor signaling transduction when administered alone, they did augment the induction of leptin receptor signaling transduction by leptin. These results demonstrate that antibodies against the extracellular domains of leptin-specific receptor enhance, but do not mimic, the ability of leptin to activate receptors. Furthermore, the enhanced leptin bioactivity observed after the intramuscular injection of anti-LEPR antibodies confirmed the occurrence of de novo leptin in the peripheral tissues and blood of treated chickens.

Accumulation of antibiotics and heavy metals in meat duck deep litter and their role in persistence of antibiotic-resistant Escherichia coli in different flocks on one duck farm.

Meat duck deep litter is considered to be an ideal environment for the evolution of bacterial antibiotic resistance if it is under poor management. The aim of this study was to characterize the accumulation of antibiotics and heavy metals in the deep litter and their role in the persistence of antibiotic resistance of Escherichia coli, and evaluate the service life of the deep litter. Samples were collected from initial, middle, and final stages of deep litter within 3 barns (zero, 4, and 8 rounds of meat duck fattening, d 34) and 9 flocks, with known consumption of antibiotics in the controlled trail. The feed and litter levels of consumed antibiotics and heavy metals were measured. E. coli (n = 147) was isolated and typed by Eric-PCR and the phylogenetic grouping technique, while minimal inhibitory concentrations of antibiotics and heavy metals were measured. This study confirmed the continuous accumulation of doxycycline and many heavy metals in the deep litter. The population of resistant certain bacteria to doxycycline (16 mg/L, 100 mg/L) or ofloxacin (8 g/mL, 50 g/mL) increased in the used deep litter (rounds 4 and 8). E. coli isolated from the 3 stages of sampling were highly resistant to ampicillin, tetracycline, florfenicol, and doxycycline. Increased resistance to ceftiofur, enrofloxacin, ofloxacin, and gentamicin were seen in the isolates from the final stage of deep litter. In addition, the percentage of isolates tolerant to zinc, copper, and cadmium and the numbers of Group-B2 isolates all increased in the used deep litter, and the isolates of each stage belonged predominantly to commensal groups. The antibiotic resistance of isolates with identical Eric-PCR patterns had improved from round 4 to 8, and differences still existed in the resistance profiles of isolates with identical Eric-PCR patterns from different barns of the same round. This study concluded that deep litter could be suitable for the evolution of bacterial antibiotic-resistance under conditions of continuous usage or accumulation of antibiotics and heavy metals without proper management.

Creating leptin-like biofunctions by active immunization against chicken leptin receptor in growing chickens.

In this study, immunization against chicken leptin receptor (cLEPR) extracellular domain (ECD) was applied to investigate leptin regulation and LEPR biofunction in growing chicken pullets. A recombinant protein (cLEPR ECD) based on the cLEPR complemenary DNA sequence corresponding to the 582nd to 796th amino acid residues of cLEPR mature peptide was prepared and used as antigen. Immunization against cLEPR ECD in growing chickens increased anti-cLEPR ECD antibody titers in blood, enhanced proportions of phosphorylated janus kinase 2 (JAK2) and served as signal transducer and activator of transcription 3 (STAT3) protein in liver tissue. Chicken live weight gain and abdominal fat mass were significantly decreased (P < 0.05), but feed intake was stimulated by cLEPR ECD immunization (P < 0.05). The treatment also upregulated the gene expression levels of lepR, AMP-activated protein kinase (AMPK), acetyl CoA carboxylase-2 (ACC2), and uncoupling protein 3 (UCP3) in liver, abdominal fat, and breast muscle (P < 0.05) but decreased fasn expression levels (P < 0.01). Apart from that of lepR, the expression of appetite-regulating genes, such as orexigenic genes, agouti-related peptide (AgRP) and neuropeptide Y (NPY), were upregulated (P < 0.01), whereas the anorexigenic gene proopiomelanocortin (POMC) was downregulated in the hypothalamic tissue of cLEPR-immunized pullets (P < 0.01). Blood concentrations of metabolic molecules, such as glucose, triglycerides, and very-low-density lipoprotein, were significantly decreased in cLEPR-immunized pullets but those of cholesterol, high-density lipoprotein, and low-density lipoprotein increased. These results demonstrate that antibodies to membrane proximal cLEPR ECD enhance cLEPR signal transduction, which stimulates metabolism and reduces fat deposition in chickens.

Construction of conditionally replicating adenovirus expressing staphylococcal enterotoxin A gene: potential usefulness for anti-tumor therapies.

OBJECTIVE: The aim of this study was to construct a conditionally replicating adenovirus pPE3-SEA expressing staphylococcal enterotoxin A (SEA) gene. MATERIALS AND METHODS: A full-length SEA gene fragment was cloned into pENTR12 plasmid to obtain a recombinant viral plasmid pENTR12-SEA. The pENTR12-SEA plasmid was co-transfected into HEK293 cells along with pPE3-ccdB, which encoded for the virus backbone, to generate recombinant adenovirus pPE3-SEA vector. Amplified pPE3-SEA vectors were purified, and viral titer was determined using the 50% tissue culture infective dose method. RESULTS: The PCR, restriction enzyme digestion, and sequence analyses proved successful construction of replicating oncolytic adenovirus pENTR12-SEA and recombinant SEA expressing oncolytic adenovirus pPE3-SEA. The viral titer was 2.5 × 1010 pfu/ml. CONCLUSIONS: We successfully constructed conditionally replicating adenovirus pPE3-SEA which can be utilized for experimental studies of tumor-targeted therapies.

Cbl-b gene silencing in splenic T lymphocytes as a therapeutic strategy to target the prostate cancer RM-1 cell tumors in immune competent mice.

OBJECTIVE: In recent years, the field of cancer immunotherapy has become a research hotspot and is currently faced with numerous challenges. The objective of this study was to assess the success of cbl-b gene silencing in splenic T lymphocytes as an immune strategy to target the murine prostate cancer RM-1 cells in vitro and solid tumors in vivo. MATERIALS AND METHODS: For this purpose, cbl-b gene-specific siRNA was designed, synthesized, and was transfected into mouse splenic T lymphocytes, followed by assessment of T cell activation, TH1 cytokine production, and in vitro cytotoxicity against RM-1 cell targets. For in vivo cytotoxicity studies, first the RM-1 tumor model was established in immune competent mice that were later tumor-injected with splenic T lymphocytes transfected with specific shRNA for cbl-b gene silencing. RESULTS: The data show that the cbl-b gene silencing in T lymphocytes resulted in an enhanced surface expression of CD69 activation marker, elevated production of interleukin (IL)-2 and interferon (IFN)-γ, and their increased cytotoxicity as effectors against RM-1 prostate cancer cells. The tumor injection with cbl-b shRNA-transfected T lymphocytes also resulted in significant reduction of the tumor size as compared with controls. CONCLUSIONS: cbl-b gene silencing strategy enhanced the immune function of T lymphocytes, increased their cytotoxic potential against RM-1 prostate cancer cells, as well as caused significant suppression of the tumor growth in immune competent mice.

Studies on enhancing embryo quantity and quality by immunization against inhibin in repeatedly superovulated Holstein heifers and the associated endocrine mechanisms.

To investigate the feasibility of improving embryo production in cattle by immunization against inhibin, both in vivo and in vitro experiments were conducted. In two experiments conducted in two autumns, 14 animals aged 14 months were immunized with a recombinant inhibin α subunit protein antigen for four times at monthly intervals, with another 14 animals of the same age served as the controls. Starting from the second immunization, all the heifers received standard superovulation treatment for three sessions, one session per month, each starting 10 days after every antigen administration. Immunization against inhibin increased number of transferable embryos (P<0.05), and high quality Grade A embryos (P<0.01) in each superovulation. Blood concentrations of FSH, estradiol, activin, and also ratio of activin to follistatin concentrations were greater in inhibin immunized than in control animals during the period of superovulatory FSH administration and animal estrous expression. Heifers immunized with inhibin also had greater concentrations of progesterone in the later diestrus period. In the second experiment, the inclusion of anti-inhibin antibody in oocyte IVM medium increased oocyte maturation rate and cleavage rate following IVF (P<0.05). These results demonstrated that inhibition of the adverse effects of inhibin on ovarian follicular development and oocyte maturation improved embryo yield, in both quantity and quality, following superovulation. These results also demonstrate that active immunization against inhibin, in conjunction with the conventional superovulation protocol, can constitute a new technique for consistent improvement of bovine embryo production in vivo; while passive immunization with anti-inhibin antibody can improve embryo production in vitro.

Immunisation against inhibin enhances follicular development, oocyte maturation and superovulatory response in water buffaloes.

This study was carried out to test the feasibility of enhancing embryo production in vivo and in vitro by immunoneutralisation against inhibin or follistatin. In Experiment 1, multi-parity buffaloes were assigned into three groups: High group (n=8), which received one primary (2mg) and two booster (1mg) vaccinations (28-day intervals) with a recombinant inhibin α subunit in 1 mL of white oil adjuvant; Low group (n=8), which received half that dose; and Control group (n=7), which received only adjuvant. Immunisation against inhibin stimulated development of ovarian follicles. Following superovulation and artificial insemination, inhibin-immunised buffaloes had more developing follicles than the Control buffaloes. The average number of embryos and unfertilised ova (4.5±0.6, n=6) in the High group was higher (P<0.05) than in the Control group (2.8±0.6, n=5) and was intermediate (4.1±0.7, n=7) in the Low group. The pooled number of transferable embryos of the High and Low groups (3.2±0.5, n=13) was also higher (P<0.05) than that (1.6±0.7, n=5) of the controls. The immunised groups also had higher plasma concentrations of activin, oestradiol and progesterone. In Experiment 2, the addition of anti-inhibin or anti-follistatin antibodies into buffalo oocyte IVM maturation medium significantly improved oocyte maturation and cleavage rates following parthenogenic activation. Treatment with anti-follistatin antibody also doubled the blastocyst yield from activated embryos. These results demonstrated that immunisation against inhibin stimulated follicular development, enhanced oocyte quality and maturation competence, yielded more and better embryos both in vivo and in vitro.

Prolactin plays a stimulatory role in ovarian follicular development and egg laying in chicken hens.

The aim of this study was to show a stimulatory role in ovarian follicle development by prolactin (PRL) in chicken hens. In experiment 1, anti-PRL antibodies were generated in hen plasma by intramuscular administrations of recombinant PRL antigen. Egg laying remained at levels lower (P < 0.05) in the PRL-immunized group than in the BSA-immunized group of hens, whereas development of incubation was depressed in the former but not the latter group. Throughout the experiment, plasma PRL concentrations were lower in the PRL-immunized hens than in non-incubating control hens; LH concentrations were similar between the PRL- and BSA-immunized hens until the end of the experiment when LH was lower in the BSA-immunized hens (P < 0.05). In experiment 2, anti-PRL receptor (PRLR) antibodies were raised in hens with the use of immunizations against recombinant PRLR extracellular domain. Immunization against PRLR initially increased the egg-laying rate when measured under the short photoperiod (12 h) but blocked the laying rate increase that occurred in the BSA-immunized control hens when the photoperiod was extended from 12 to 16 h. The development of incubation behavior was not affected by immunization against PRLR nor was plasma PRL or LH concentration. In experiment 3, when the egg-laying rate was depressed in PRL immunization hens, developmental speed of large white follicles was found to be slower than in the BSA-immunized control hens (P < 0.05). These results indicate that immunization against PRL slows down ovarian follicular development and reduces hen egg-laying performance, suggesting that PRL plays a stimulatory role in ovarian follicular development in chicken hens.

Immunization against inhibin enhances both embryo quantity and quality in Holstein heifers after superovulation and insemination with sex-sorted semen.

The objective was to investigate the feasibility of improving embryo yield in superovulated cows following insemination with sex-sorted semen by prior immunization against inhibin. Twenty-eight heifers were allocated into three groups: High (n=10), Low (n=10), and Control (n=8). The High group received one primary (1mg) and two booster (0.5mg) vaccinations (28-d intervals) with a recombinant inhibin alpha-subunit in 1 mL of white oil adjuvant, whereas the Low group received half that dose, and the Control group received only adjuvant. After the last immunization, all heifers underwent a standard superovulation treatment (decreasing doses of pFSH for 4d), followed by two AI with 2 x 10(6) sex-sorted semen after the onset of estrus. Inhibin-immunized heifers had higher (P<0.01) plasma antibody titres, and an earlier onset of estrus (P<0.05) than Control heifers. The total number of embryo/ova, transferable, and grade 1 embryos in the High group (15.4+/-1.9, 5.7+/-0.7, and 3.8+/-1.0, respectively) was significantly greater than that of the Control group (9.1+/-1.2, 3.1+/-0.5, and 0.6+/-0.2), but was intermediate (P>0.05) in the Low group (13.0+/-2.3, 4.4+/-0.7, and 1.2+/-0.3). There were no significant differences among groups in number of unfertilized ova and degenerated embryos. The High group also had higher (P>0.05) plasma progesterone concentrations on the day of embryo collection. In conclusion, immunization against inhibin improved both embryo quantity and quality following superovulation and insemination with sex-sorted semen.

Enhancing embryo yield in superovulated holstein heifers by immunization against inhibin.

Eight heifers, aged 16-17 months and showing normal oestrous cycles, were immunized against a recombinant porcine inhibin alpha subunit immunogen, together with another 10 heifers of the same age as controls and treated with placebo immunogen. Primary (1 mg immunogen) and two booster (0.5 mg immunogen each) immunizations were administered at 28-day intervals. Ten days after the second booster immunization, both groups of heifers underwent a superovulation treatment. Each animal was given an intravaginal progesterone releasing sponge, which was withdrawn 7 days following an i.m. injection of 0.5 mg cloprostenol. Heifers were treated with FSH for 4 days and artificially inseminated after oestrus occurred. The embryos were flushed and evaluated 7 days after insemination. Immunization significantly (p < 0.01) increased blood antibody titres against recombinant porcine inhibin alpha subunit, from pre-immunizaion and control values of approximately 0.06 of ELISA 450 nm reading to 0.6 to 0.7 after two or three immunizations. The immunized heifers produced on average 15.8 +/- 2.8 embryos, significantly (p < 0.05) higher than the yield of 8.3 +/- 1.5 in the controls. The number of transferable embryos were non-significantly higher in immunized than in control heifers (9.6 +/- 3.1 vs 5.8 +/- 1.6, p > 0.05). The peak plasma oestradiol concentrations were significantly higher in immunized than in control heifers, both immediately after FSH treatment and 20 days thereafter. Plasma P4 concentrations after superovulation were in the range of 20 ng / ml in the immunized heifers, significantly (p < 0.05) higher than the values approximately 15 ng / ml in control heifers. These results indicated that prior immunization against inhibin alpha subunit stimulated production of antibodies against inhibin, which enhanced follicular developmental response to superovulation and lead to higher yield of total and transferable embryos. Therefore immunization combined with the conventional superovulatory gonadotrophin treatment, can be a simple and efficient method to produce low cost bovine embryos.

Offspring from heterotopic transplantation of newborn mice ovaries.

This study is aimed at investigating the developmental potential of the primordial follicles from ovaries of newborn mice after cryopreservation in liquid nitrogen for long-term storage, thawing, and heterografting into the kidney capsules of ovariectomized adult female mice. After stimulation of recipient mice with pregnant mare serum gonadotropin on day-19 after heterografting, the primordial follicles of the transplanted ovaries could develop into antral follicles. When the oocyte-cumulus cell complexes were retrieved from these antral follicles, they could mature after in vitro culture for 16­17 h. After in vitro fertilization, the rates of embryos derived from these oocytes that developed into the two-cell stage and the blastocyst stage after 16­17 h and after day-4, respectively,in the culture medium were 55.40% (55/107) and 9.09% (5/55),respectively. In the ovarian transplantation groups, no pups were derived from the 410 embryos that were transferred into 10 pseudopregnant mothers at the pronuclear stage. However,of the 10 surrogate mothers in whom 570 embryos were transferred at the two-cell stage, four achieved pregnancy and gave birth to 20 live offspring. These results demonstrated that primordial follicles in newborn mice ovaries were capable of sustaining their developmental potential after freezing and thawing. Once transplanted into the kidney capsules of ovariectomized adult female mice, these primordial follicles could develop and respond to gonadotropin stimulation and reach the antral stage; further, live offspring could be derived from these follicles.