Turkey ovarian tissue transplantation: effects of surgical technique on graft attachment and immunological status of the grafts, 6 days post-surgery.

Biobanked poultry ovaries can be revived via transplantation into a recipient female, which upon maturity will produce donor-derived progeny. Previously, a large portion of these recipients also produced recipient-derived progeny, making them gonadal chimeras. These were potentially created when portions of the recipient's ovary were inadvertently left behind. Completely removing the recipient ovary would solve this problem; however, leaving a portion of the recipient's ovary may have inadvertently increased the transplant attachment rate by providing a damaged area for attachment. To test this hypothesis in the turkey, we removed various portions (33-100%) of recipient ovarian tissue and determined the transplant attachment rate. Furthermore, the use of the abdominal air sac membrane as an additional anchoring point was tested. The overall attachment rate of transplants was 91% (27/30), while the average size of the transplants was 4.2 ± 0.6 mm2, 6 d postsurgery. There was no difference (P > 0.05) in the attachment rates, or transplant size between groups with varying amounts of recipent tissue removed, or by using the abdominal air sac membrane as an anchor. Finally, the immunological status of the grafts were evaluated by analyzing the presences of CD3 and MUM-1 (T and B cell markers). This showed that all transplants were infiltrated by large numbers of T and B cells. Shown by a high (P ≤ 0.001) percentage of CD3-positive immunostained cytoplasmic area (49.78 ± 3.90%) in transplants compared to remnant recipient tissue (0.30 ± 0.10%), as well as a high (P ≤ 0.001) percentage of MUM-1-positive immunostained nuclear area (9.85 ± 1.95%) in transplants over remnant recipient tissues (0.39 ± 0.12%). From this study we would recommend removing the entire recipient ovary, and not covering the transplants with the abdominal air sac membrane, to prevent gonadal chimeras. The high levels of lymphocytes within the grafts indicate possible tissue rejection, which could be overcome via immunosuppression with or without histocompatibility matching between donors and recipients.

In ovo culturing of turkey (Meleagris gallopavo) ovarian tissue to assess graft viability and maturation of prefollicular germ cells and follicles.

Biobanking of turkey ovarian tissue appears to be the most cost-effective method for the long-term preservation of female genetics. However, to ensure the successful transplantation of biobanked ovarian tissue for breed or line revival, the transplantation and development of fresh ovarian tissue must be evaluated. To assess transplantability, ovaries from poults 1 to 15 days posthatch (dph) were cultured in ovo in chicken eggs for 6 d and compared with the equivalent fresh tissue. The viability of cultured ovarian tissue was evaluated visually, whereas the level of late-stage apoptosis was measured via the TUNEL assay. In addition, the diameter and density of prefollicular germ cells and follicles (primordial and primary) were measured to assess maturation. Results showed that all cultured grafts (74/74), on surviving chicken chorioallantoic membrane, were viable with low levels (0.8 ± 0.1%) of late-stage apoptosis. The diameter of prefollicular germ cells in cultured ovaries from poults at 5 and 7 dph were larger (P < 0.002) than that of their preculture counterparts but were not able to reach their in vivo size. No significant follicular growth was observed in ovaries cultured in ovo; however, prefollicular germ cell density was over 4-fold greater in ovaries cultured from 7 dph poults (81,030 ± 17,611/mm3) than in their in vivo counterpart (16,463 ± 6,805/mm3). Interestingly, cultured ovaries from all other ages displayed equal or lower (P ≤ 0.05) prefollicular germ cell densities than their in vivo counterparts. Cultured ovaries from poults at 5 and 7 dph also exhibited an increase (P ≤ 0.05) in follicle density compared with their preculture counterparts; whereas, cultured ovaries from 15 dph poults had decreased densities (P < 0.001) compared with their preculture counterparts. This study demonstrated that, although age of ovarian tissue cultured in ovo did not affect the overall viability, 7 dph ovaries appeared to have a better cellular morphology after culturing in ovo than other ages. In addition, we also demonstrated for the first time that avian follicles can form during tissue culturing in ovo.

Modeling life-time energy partitioning in broiler breeders with differing body weight and rearing photoperiods.

Understanding energy partitioning in broiler breeders is needed to provide efficiency indicators for breeding purposes. This study compared 4 nonlinear models partitioning metabolizable energy (ME) intake to BW, average daily gain (ADG), and egg mass (EM) and described the effect of BW and rearing photoperiod on energy partitioning. Ross 708 broiler breeders (n = 180) were kept in 6 pens, controlling individual BW of free run birds with precision feeding stations. Half of the birds in each chamber were assigned to the breeder-recommended target BW curve (Standard) or to an accelerated target BW curve reaching the 21-week BW at week 18 (High). Pairs of chambers were randomly assigned to 8L:16D, 10L:14D, or 12L:12D rearing photoschedules and photostimulated with 16L:8D at week 21. Model [I] was: MEId = a × BWb + c × ADG × BWd + e × EM + Îµ, where MEId = daily ME intake (kcal/day); BW in kg; ADG in g/day; EM in g/day. Models [II-IV] were nonlinear mixed versions of model [I] and included individual [II], age-related [III], or both individual and age-related [IV] random terms to explain these sources of variation in maintenance requirement (a). Differences were reported as significant at P ≤ 0.05. The mean square error was 2,111, 1,532, 1,668, and 46 for models [I-IV] respectively, inferring extra random variation was explained by incorporating 1 or 2 random terms. Estimated ME partitioned to maintenance [IV] was 130.6 ± 1.15 kcal/kg0.58, and the ME requirement for ADG and EM were 0.63 ± 0.03 kcal/g/kg0.54 and 2.42 ± 0.04 kcal/g, respectively. During the laying period, maintenance estimates were 124.2 and 137.4 kcal/kg0.58 for standard and high BW treatment, and 130.7, 132.2, and 129.5 kcal/kg0.58 for the 8L:16D, 10L:14D, or 12L:12D treatments, respectively. Although hens on the standard BW treatment with a 12L:12D rearing photoschedule were most energetically conservative, their reproductive performance was the poorest. Model IV provided a new biologically sound method for estimation of life-time energy partitioning in broiler breeders including an age-related random term.

Germ cell dynamics during nest breakdown and formation of the primordial follicle pool in the domestic turkey (Meleagris gallopavo).

This study determined, for the first time, the different subpopulations of germ cells and stereological changes within the cortex of the functional left ovary during germ cell nest breakdown, and formation of the primordial follicle pool in the domestic turkey. This was accomplished by measuring the size, density, and count of prefollicular germ cells and primordial follicles in turkey poults between 1 and 35 days posthatch (dph). The percent volume (PV) of germ cells and follicles within the cortex was also calculated as a means of validating the counting technique. The total percent volume of germ cells and primordial follicles within the cortex ranged between 42 and 84%, suggesting that the counting technique was valid. Our findings show that before germ cell nest breakdown (5 dph), there were roughly 1,000,000 prefollicular germ cells within the cortex of the left ovary and that germ cell nest breakdown initiated between 5 and 7 dph, characterized by a decrease (P ≤ 0.001) in prefollicular germ cell density and the subsequent appearance of primordial follicles. Nest breakdown is followed on day 9 by the first increase (P ≤ 0.05) in size of prefollicular germ cells. These cells continue to grow throughout nest breakdown. The majority (>90%) of germ cell nest breakdowns concluded by 15 dph; although, the primordial follicle pool was not fully established until 35 dph, as determined by a total lack of prefollicular germ cells. At this point, the pool was comprised of an estimated 60,000 primordial follicles and shows that during nest breakdown and follicle pool formation, ∼94% of germ cells were lost. This 94% decrease in the number of germ cells during nest breakdown in the turkey is comparable to the domestic chicken but is greater than the average two-thirds which are lost in mammalian species.

Impact of feeding microalgae (Aurantiochytrium limacinum) and co-extruded mixture of full-fat flaxseed as sources of n-3 fatty acids to ISA brown and Shaver white breeders and progeny on pullet skeletal attributes at hatch through to 18 weeks of age.

Impact of feeding n-3 fatty acids (FA) to ISA brown and Shaver white breeders and their progeny on bone development in pullets was investigated. Breeders were fed Control (CON); CON + 1% microalgae (DMA: Aurantiochytrium limacinum) as the source of docosahexaenoic acid; and CON + 2.6% of a co-extruded mixture of full-fat flaxseed (FFF) and pulses mixture as source of α-linolenic acid. Test diets (DMA and FFF) were balanced for total n-3 FA and n-6: n-3 FA ratio. Samples of day-old progeny were euthanized for bone mineral content (BMC) and tibia collagen type II. The remaining pullets were fed posthatch treatments as follows: from breeder CON: CON (CON-CON), DMA (CON-DMA), and FFF (CON-FFF), from breeder DMA: CON (DMA-CON) and DMA (DMA-DMA) and from breeder FFF: CON (FFF-CON) and FFF (FFF-FFF). A total of 60 pullets per posthatch diets were reared in cages (12 pullets/cage, n = 5) with free access to feed and water, bled at 6, 12, and 18 wk of age (WOA) for bone turnover markers and necropsied at 18 WOA for tibia and femur samples. Day-old pullets from breeder fed CON had greater BMC (P < 0.001) relative to those from breeders fed other diets. There was strain and diet interaction (P ≤ 0.024) on tibia breaking strength (TBS) and tibia cortical ash concentration at 18 WOA such that diet responses were only observed in Shaver white pullets. In this context, TBS of DMA-DMA and FFF-FFF was greater than for pullets originating from CON breeder, and the cortical ash weight of DMA-DMA and FFF-FFF pullets was 23.8 and 20.2%, respectively, higher than for CON-CON pullets. In conclusions, the strain effects were strong on tibia attributes on 18-week-old pullets. Breeder feeding of n-3 FA was more effective when concomitant with posthatch feeding of n-3 FA in supporting the skeletal strength and cortical bone development in Shaver white pullets. Further investigations are warranted to establish the impact these strategies on skeletal health during laying cycle.

Post-photostimulation energy intake accelerated pubertal development in broiler breeder pullets.

The effect of ME intake (MEI) on the reproductive system was evaluated. Ross 308 broiler breeder pullets (n = 140) were assigned to 2 treatments from 22 to 26 wk of age: (1) Low-energy diet fed restricted (2,807 kcal/kg, low MEI) and (2) high-energy diet fed unrestricted (3,109 kcal/kg, high MEI). Daylength was increased from 8 to 14 h at 22 wk of age with a light intensity of 30 lux. Daily palpation was used to detect sexual maturity via the presence of a hard-shelled egg in the shell gland. Expression of gonadotropin releasing hormone-I (GnRH) and gonadotropin inhibitory hormone (GnIH) genes in the hypothalamus and GnRH receptor (GnRH-RI) and GnIH receptor (GnIH-R) genes in the anterior pituitary gland of each pullet was evaluated from 22 to 26 wk of age using quantitative real time-PCR. Blood samples were taken weekly and luteinizing hormone (LH), follicle stimulating-hormone (FSH), and 17-beta-estradiol (E2) determined using commercial ELISA kits. Carcass samples were used for determination of CP and fat content. Data were analyzed using the MIXED procedure in SAS, and differences were reported where P ≤ 0.05. High MEI treatment pullets had 2.3-fold higher GnRH and 1.8-fold higher GnRH-RI mRNA levels than low MEI pullets. MEI affected neither expression of GnIH and GnIH-R nor carcass protein content. For high MEI (489 kcal/D) and low MEI treatments (258 kcal/D), respectively, from 22 to 26 wk of age (P ≤ 0.05), LH concentration was 3.05 and 1.60 ng/mL; FSH concentration was 145 and 89.3 pg/mL; E2 concentration was 429 and 266 pg/mL, and carcass lipid was 13.9 and 10.3%. The onset of lay for pullets in the high MEI treatment advanced such that 100% had laid by 26 wk of age compared with 30% in the low MEI treatment. We concluded that higher MEI advanced the activation of the hypothalamic-pituitary-gonadal axis and also increased body lipid deposition, and moreover, stimulated reproductive hormone levels which overall accelerated puberty in broiler breeder pullets.

A model of pre-pubertal broiler breeder estradiol-17ß levels predicts advanced sexual maturation for birds with high body weight or short juvenile day-length exposure.

As broiler breeders face increased reproductive challenges specifically related to overfeeding, a clear understanding of the physiological effects of BW and rearing photoperiod on reproductive development is needed. The objective was to use mathematical models to compare plasma estradiol-17ß (E2) concentration to characterize the effect of BW and rearing photoperiod on E2 levels. A 2 × 3 factorial arrangement of treatments was used. Hens (n = 180) were fed with a precision feeding system to allocate feed individually to achieve the breeder-recommended BW curve (Standard) or to a BW curve reaching the 21 wk target at 18 wk (High). Hens were on 8L:16D, 10L:14D, or 12L:12D photoschedules during rearing and were photostimulated at 21 wk. Age at first egg (AFE) was recorded. Plasma E2 levels were determined weekly between week 20 and 28. Two modified Gompertz models described E2 level as a function of (a) chronological or (b) physiological (relative to AFE) age. Timing of E2-inflection point was compared between models and treatments. Differences were reported as significant at P ≤ 0.05. The chronological age model inferred that High BW reduced the duration between the E2-inflection point and AFE, whereas the physiological age model inferred that High BW only reduced the duration between photostimulation and the E2-inflection point. Hens on the Standard BW treatment had a longer period between photostimulation and the E2-inflection point compared to hens on the High-BW treatment (11.03 vs. 1.50 wk, respectively, based on physiological age). Hens on the 12L:12D photoschedule had a longer period between photostimulation and the E2-inflection point compared to hens on the 8L:16D or 10L:14D photoschedule, both in the Standard and High BW (28.91 vs. 1.78 and 2.40 wk, 2.65 vs. 0.93 and 0.94 wk, respectively, based on physiological age). The described methodology and results provide quantitative insight into E2 dynamics and serves as a model for future endocrinological studies in poultry reproduction.

The effect of rearing photoperiod on broiler breeder reproductive performance depended on body weight.

Body weight (BW) and rearing photoperiod are important factors affecting sexual maturation rate and reproductive performance in broiler breeders. The current experiment used a 2 × 3 factorial arrangement of treatments to study the interaction between BW and rearing photoperiod on reproductive performance in group housed broiler breeder hens, while minimizing variation in BW. Hens (n = 180) were fed with a precision feeding system to allocate feed individually to achieve the breeder-recommended target curve (Standard) or to a target curve that reached the 21 wk BW at 18 wk (High). Hens were on 8L:16D, 10L:14D, or 12L:12D photoschedules during rearing and were photostimulated at 21 wk with a 16L:8D photoschedule. Sexual maturity (defined as age at first egg) and individual egg production to 55 wk were recorded. At 55 wk, proportional weights of individual body components were determined by dissection. Differences were reported as significant at P ≤ 0.05. A significant interaction between BW and rearing photoschedule affected age at sexual maturity and egg production. In the High BW treatment, age at sexual maturity did not differ between hens under the 8L:16D and 10L:14D photoschedules (173 vs. 172 d, respectively). In the Standard BW treatment, the 12L:12D rearing photoperiod delayed sexual maturity compared with the 8L:16D rearing photoperiod (266 vs. 180 d, respectively). All hens on the High BW treatment laid at least 1 egg before the end of the experiment. Conversely, 3.3, 18.1, and 37.6% of Standard BW hens on the 8L:16D, 10L:14D, and 12L:12D photoschedules, respectively, never commenced egg production. At the end of the experiment, proportional breast weight was higher and proportional fatpad weight was lower in Standard compared to High BW hens (25.8 vs. 27.5% and 2.4 vs. 1.5% of BW, respectively). We conclude that increased BW partially counters the effect of longer photoschedules on sexual maturity in broiler breeders and that dissipation of the photorefractory state depends on BW.

Early photostimulation at the recommended body weight reduced broiler breeder performance.

To synchronize the onset of sexual maturity in the face of high BW variation, the age at photostimulation has been increasing in the broiler breeder industry. This experiment studied the effects of increased BW and earlier photostimulation on broiler breeder reproductive performance where within-treatment BW uniformity was very high. The experiment tested BW and age at photostimulation treatments in a 2 × 2 factorial arrangement. Hens (n = 120) were fed with a precision feeding system to allocate feed individually following the breeder-recommended target BW (Standard) or to a 22% heavier target BW curve reaching the Standard 21 wk BW at 18 wk (High). Hens were photostimulated at either 18 wk (18WK) or 21 wk (21WK) with a 16L:8D photoschedule. Age at first egg (AFE) and individual egg production to 55 wk were recorded. Differences were reported as significant if P ≤ 0.05. The AFE was decreased and maturation interval between photostimulation and AFE was shorter for hens on the High BW treatment compared to the Standard BW treatment (178.1 vs. 194.7 d and 41.8 vs. 58.2 d, respectively). Hens on the 21WK treatment had a decreased AFE compared to the 18WK treatment (177.0 d vs. 195.9 d) and their maturation interval was shorter (30.0 d vs. 69.9 d). The CV for AFE was higher in the 18WK treatment compared to the 21WK treatment (28.2% vs. 11.2%). Total egg production was higher for hens on the High BW treatment compared to the Standard BW treatment (129.4 vs. 92.8, respectively). Total egg production was higher for hens on the 21WK treatment compared to the 18WK treatment (138.4 vs. 83.8, respectively). Egg weight of Standard BW × 18WK hens was lower compared to High BW × 18WK hens. Current recommended breeder BW may be too low for optimal sexual maturation after photostimulation. It is concluded that even when BW variation is minimized, photostimulation at 18 wk of age is not recommended.

Red light is necessary to activate the reproductive axis in chickens independently of the retina of the eye.

Photoperiod is essential in manipulating sexual maturity and reproductive performance in avian species. Light can be perceived by photoreceptors in the retina of the eye, pineal gland, and hypothalamus. However, the relative sensitivity and specificity of each organ to wavelength, and consequently the physiological effects, may differ. The purpose of this experiment was to test the impacts of light wavelengths on reproduction, growth, and stress in laying hens maintained in cages and to determine whether the retina of the eye is necessary. Individual cages in 3 optically isolated sections of a single room were equipped with LED strips providing either pure green, pure red or white light (red, green, and blue) set to 10 lx (hens levels). The involvement of the retina on mediating the effects of light wavelength was assessed by using a naturally blind line (Smoky Joe) of chickens. Red and white lights resulted in higher estradiol concentrations after photostimulation, indicating stronger ovarian activation, which translated into a significantly lower age at first egg when compared with the green light. Similarly, hens maintained under red and white lights had a longer and higher peak production and higher cumulative egg number than hens under green light. No significant difference in BW gain was observed until sexual maturation. However, from 23 wk of age onward, birds exposed to green light showed higher body growth, which may be the result of their lower egg production. Although corticosterone levels were higher at 20 wk of age in hens under red light, concentrations were below levels that can be considered indicative of stress. Because no significant differences were observed between blind and sighted birds maintained under red and white light, the retina of the eye did not participate in the activation of reproduction. In summary, red light was required to stimulate the reproductive axis whereas green light was ineffective, and the effects of stimulatory wavelengths do not appear to require a functional retina of the eye.

Development, validation, and utilization of a novel antibody specific to the type III chicken gonadotropin-releasing hormone receptor.

Two gonadotropin-releasing hormone receptors (GnRH-Rs) have been characterized in chickens to date: cGnRH-R-I and cGnRH-R-III, with cGnRH-R-III being the predominant pituitary form. The purpose of the present study was to first validate a novel antibody for the specific detection of cGnRH-R-III and second, using this antibody, detect changes in cGnRH-R-III protein levels in the pituitary gland of male and female chickens during a reproductive cycle. The localization of cGnRH-R-III within the anterior pituitary gland was also determined. Western blotting of pituitary extracts and transiently transfected COS-7 cell lysates revealed that our antibody is highly specific to cGnRH-R-III protein. Similarly, when used in immunocytochemistry, this antibody specifically detects cells expressing cGnRH-R-III and not cGnRH-R-I. Western blot analyses of chicken pituitary gland homogenates show that cGnRH-R-III protein levels are significantly greater in sexually mature birds than in immature birds or birds at the end of a reproductive cycle (P < 0.0001). A similar pattern was observed for both males and females. Additionally, the antibody was able to detect cGnRH-R-III in cells along the periphery of the cephalic and caudal lobes of the anterior pituitary where the cells containing the gonadotropins are located. In summary, we successfully validated a novel antibody to cGnRH-R-III and showed levels of cGnRH-R-III protein in the pituitary fluctuate with respect to the reproductive status in both male and female chickens.

Use of dietary thyroxine as an alternate molting procedure in spent turkey breeder hens.

In the turkey industry, molting is traditionally achieved by reducing photoperiod and withdrawing feed and water for several days. Although it is the most effective method, this practice is discouraged in Canada and alternative strategies need to be established. Thyroid hormone levels naturally change during molt, and dietary thyroxine (T4) supplementation was previously shown to induce molt in chickens. This study aimed at evaluating the effectiveness of supplemental dietary T4 in inducing molt in spent turkey breeder hens. One hundred twenty 75-wk-old hens were randomly divided into 4 groups (5 floor pens/replicates, 5 hens each) with the control group kept under a 14-h photoperiod and fed a breeder's diet throughout, whereas hens from the 3 other groups were supplemented with 40 ppm (45.76 mg/kg) T4 for 10 d. One treatment group was maintained under 14 h of light and fed a breeder's diet, whereas the 2 others were subjected to a drop in photoperiod to 6 h during or after supplementation and then were fed a maintenance diet. Egg production, feed intake, BW, molt, and plasma levels of T4, prolactin, and luteinizing hormone were measured. All treated hens ceased laying by d 20; however, several individuals spontaneously returned to lay when left on 14 h of light, suggesting incomplete involution of the reproductive tract. Supplementation significantly reduced feed consumption and induced rapid BW loss. All hens returned to their initial weight by the end of the experiment. Most treated hens initiated molt by d 8 of supplementation and all completed molt by d 37. Plasma T4 in treated hens increased significantly by d 3 (P < 0.05) and remained significantly higher than in controls until d 9 (P < 0.01). Levels returned to initial values by d 35. Prolactin levels did not appear to be influenced by T4 but were mainly dependent on photoperiod and reproductive stage, whereas luteinizing hormone levels remained low throughout. In summary, dietary supplementation with 40 ppm (45.76 mg/kg) T4 was successful in inducing molt in turkey breeder hens. However, dropping the photoperiod was necessary to completely reset the reproductive system.

Expression patterns of the prolactin receptor gene in chicken lymphoid tissues during embryogenesis and posthatch period.

Prolactin (PRL) is a pituitary hormone with multiple homeostatic roles among vertebrates. Although it has mainly been studied in relation to its role during the initiation and maintenance of incubation behavior in avian species, it has also been shown to act on the immune system. In this study, levels of PRL receptor (PRLR) mRNA were quantified by real-time PCR, and tissue expression was localized by in situ hybridization in primary and secondary lymphoid organs. Prolactin receptor was shown to be expressed in the bursa follicles, thymus lobules, and splenic pulp at all stages of development examined. Levels of PRLR expression were consistently higher in the bursa of Fabricius when compared with other lymphoid organs, suggesting that PRL acts primarily on bursal development. Furthermore, levels of PRLR mRNA appeared to fluctuate during embryogenesis, with a significant increase observed at embryonic day 19 in the bursa, at 7 d of age in the thymus, and on hatching day in the spleen. Thus, PRL might play an important role during the development of the immune system in chickens.

Ontogenesis of the expression of prolactin receptor messenger ribonucleic acid during late embryogenesis in turkeys and chickens.

Changes in circulating levels of prolactin (PRL) and tissue content of PRL receptor (PRLR) messenger RNA (mRNA) in the liver, pancreas, kidney, and gonads (testis/ovary) were measured in turkey and chicken embryos from embryonic day (ED) 21 or ED15, respectively, to 1 d after hatch by real-time PCR. There were no differences between the sexes in chickens or turkeys. Both species had very similar patterns of PRL release and expression of PRLR mRNA, and no major differences were observed between turkey or chicken embryos. Plasma levels of PRL increased from low levels during the last week of embryonic development and were at significantly higher levels (about 4-fold) by 1 d after hatch. Similarly, in all tissues the content of PRLR mRNA was minimal at the outset and increased to reach maxima about the time of hatch. In both species, the highest levels of transcript were observed in the kidney followed by the gonad, liver, and pancreas. The tissue content of PRLR was correlated (0.6 to 0.8 dependent on the tissue) to circulating levels of PRL, which suggested that PRL may be associated with an increase in its receptor number around the time of hatch. Because levels of PRL and tissue content of PRLR mRNA increased around the time of hatch, this suggests that these tissues may be targets for PRL and may be involved in the physiologic changes occurring in embryos around the time of hatching.

Development of a real-time (Q) PCR assay to measure variation in expression of prolactin receptor mRNA in the hypothalamus and pituitary gland during late embryogenesis in turkeys and chickens.

Changes in levels of PRLR mRNA in the pituitary gland and hypothalamus of chickens and turkeys from embryonic day (ED) 15 and ED21 to 1 day post-hatch, respectively, were measured by real-time PCR. In both species, PRLR mRNA increased from low levels during the last week of ED to reach maxima at the peri-hatch period. Similarly, circulating levels of PRL also increased during this interval and were highly correlated with levels of the PRLR mRNA in both the pituitary gland and hypothalamus. This suggests that PRL was up-regulating its receptor. In support of this, stimulation of the turkey pituitary gland with VIP on ED24 resulted in a 4- and 3-fold increase in PRL and PRLR, respectively. Since VIP had no direct effect on the levels of PRLR transcript in the hypothalamus, it is likely that VIP is acting indirectly through increased PRL to up-regulate the number of receptors.

Use of a vascular access port for the measurement of pulsatile luteinizing hormone in old broiler breeders.

Techniques used to measure circulating hormone concentrations in avian species over extended periods routinely involve cannulation or multiple venipunctures under physical restraint, resulting in sepsis and stress. We adapted a method for serial blood sampling in chickens using a vascular access port (VAP) surgically implanted under the skin of the neck and connected to a catheter inserted in the right jugular vein. The system was used to measure circulating luteinizing hormone (LH) profiles in six, 21-mo-old broiler breeders at the end of their laying period. The VAP were implanted under general anesthesia, and, after a period of recovery, serial blood samples (every 10 min for 6 h) were collected using an extension line connected to a push-pull system. Birds were unrestrained and had free access to food and water. Red blood cells were recovered by centrifugation, reconstituted in saline solution, and returned to the donor bird through the VAP once every 90 min. Luteinizing hormone levels were subsequently measured in plasma by radioimmunoassay. With the exception of 1 hen that developed valvular endocarditis, no sign of disease or infection was observed throughout the study, and the VAP remained functional in all birds for at least 3 mo. Thus, our results suggest that VAP are a safe, reliable, and less stressful technique for serial blood sampling and long-term studies. Radioimmunoassay results revealed that in old birds, circulating LH levels followed a pulsatile pattern, with pulse amplitudes ranging from 1.35 to 2.02 ng/mL and pulse frequencies ranging from 5 to 6 peaks per 6 h. Although not significant, amplitude of LH pulses in out-of-lay hens appeared to be lower than in laying hens.

Two novel mutations in the gonadotropin-releasing hormone receptor gene in Brazilian patients with hypogonadotropic hypogonadism and normal olfaction.

Several point mutations in the GnRH receptor gene have been described in an autosomal recessive form of congenital isolated hypogonadotropic hypogonadism (HH). We investigated 17 Brazilian patients (10 males and 7 females) from 14 different families, with HH and normal olfaction. The diagnosis of HH was based on absent or incomplete sexual development after 17 yr of age associated with low or normal levels of LH in both sexes and low levels of testosterone in males and of estradiol in females. All patients presented with a normal sense of smell in an olfactory specific test. The coding region of the GnRH receptor gene was amplified by PCR and directly sequenced. A novel missense mutation, Arg(139)His, located in the conserved DRS motif at the junction of the third transmembrane and the second intracellular loop of the GnRH receptor was identified in the homozygous state in one female with complete HH. The Arg(139)His mutation completely eliminated detectable GnRH-binding activity and prevented GnRH-induced stimulation of inositol phosphate accumulation in vitro. In another family, a new compound heterozygous mutation (Asn(10)Lys and Gln(106)Arg) was identified in four siblings (two males and two females) with partial HH. The Gln(106)Arg mutation, located in the first extracellular loop, has been previously described, and in vitro analysis indicated that the mutant receptor was able to bind GnRH, but with a reduced affinity. The Asn(10)Lys mutation in the extracellular amino-terminal domain of the receptor also reduced the affinity for GnRH in vitro. In this family we also identified a previously described silent polymorphism at amino acid residue 151 in the second intracellular loop that segregated with the two inactivating mutations of the GnRH receptor. This polymorphism was also found in two unrelated patients with sporadic HH without GnRH receptor loss of function mutations. No mutations were identified in the remaining cases. A good correlation between genotype and phenotype was found in our patients. The woman, who is homozygous for the completely inactivating Arg(139)His mutation, has complete HH with undetectable serum basal LH and FSH levels that failed to respond to GnRH stimulation. In addition, the affected patients who are compound heterozygotes for the Asn(10)Lys/Gln(106)Arg mutations, have partial HH with low serum basal LH levels that were responsive to GnRH stimulation. No clinical or hormonal differences were found between HH patients with and without mutations in the GnRH receptor gene, indicating that these data do not contribute to the identification of HH patients with GnRH receptor mutations. In conclusion, we report the first naturally occurring mutation within the conserved DRS motif of the GnRH receptor in a female with complete HH and a novel compound heterozygous mutation (Asn(10)Lys and Gln(106)Arg) in a family with partial HH, increasing the repertoire of the inactivating mutations of the GnRH receptor.

Prevalence, phenotypic spectrum, and modes of inheritance of gonadotropin-releasing hormone receptor mutations in idiopathic hypogonadotropic hypogonadism.

Mutations in the GnRH receptor (GNRHR) have been described as a cause of reproductive failure in a subset of patients with idiopathic hypogonadotropic hypogonadism (IHH). Given the apparent rarity of these mutations, we set out to determine the frequency and distribution of GNRHR mutations in a heterogeneous population of patients with IHH who were well characterized with respect to diagnosis, phenotype, and mode of inheritance and to define their distribution within the receptor protein. One hundred and eight probands with IHH were screened for mutations in the coding sequence of GNRHR. Forty-eight of the 108 patients had a normal sense of smell, whereas the remaining 60 had anosmia or hyposmia (Kallmann syndrome). Exon segments in the GNRHR were screened for mutations using temperature gradient gel electrophoresis, and all mutations were confirmed by direct sequencing. Five unrelated probands (3 men and 2 women), all normosmic, were documented to have changes in the coding sequence of the GNRHR. Two of these probands were from a subgroup of 5 kindreds consistent with a recessive mode of inheritance, establishing a GNRHR mutation frequency of 2 of 5 (40%) in patients with normosmic, autosomal recessive IHH. The remaining 3 probands with GNRHR mutations were from a subgroup of 18 patients without evidence of familial involvement, indicating a prevalence of 3 of 18 (16.7%) in patients with sporadic IHH and a normal sense of smell. Among the five individuals bearing GNRHR mutations, a broad spectrum of phenotypes was noted, including testicular sizes in the male that varied from prepubertal to the normal adult male range. Three probands had compound heterozygous mutations, and two had homozygous mutations. Of the eight DNA sequence changes identified, four were novel: Thr(32)Ile, Cys(200)Tyr, Leu(266)Arg, and Cys(279)TYR: COS-7 cells transiently transfected with complementary DNAs encoding the human GNRHR containing each of these four novel mutations failed to respond to GnRH agonist stimulation. We conclude that 1) the spectrum of phenotypes in patients with GNRHR mutations is much broader than originally anticipated; 2) the frequency of GNRHR mutations may be more common than previously appreciated in familial cases of normosmic IHH and infrequent in sporadic cases; and 3) functional mutations of the GNRHR are distributed widely throughout the protein.

Quantification of prolactin messenger ribonucleic acid, pituitary content and plasma levels of prolactin, and detection of immunoreactive isoforms of prolactin in pituitaries from turkey embryos during ontogeny.

The content of prolactin mRNA as well as total prolactin content and type of isoforms of prolactin were measured in single pituitary glands from turkey embryos and poults. Levels of mRNA and pituitary content of prolactin remained low until 5 days before hatching, while plasma concentrations remained low until 2 days before hatching. Levels of prolactin mRNA then increased until the day of hatch, stayed stable during the 3 first days of age, and significantly increased until 2 wk of age. Similar changes were observed in pituitary content and plasma levels of prolactin. Two immunoreactive bands of apparent molecular masses of 24 and 27 kDa, corresponding to the nonglycosylated and glycosylated form of prolactin, respectively, were visualized on Western blots. In pituitary glands from embryos at 22 days of incubation, 31.5% of the protein was glycosylated, whereas in embryos at 27 days of incubation and poults at 1 and 7 days of age, 48.6%, 48.0%, and 56. 0% of prolactin was glycosylated, respectively. The results indicate that the increases in the synthesis and the release of prolactin occur mainly around and after the time of hatching in the turkey embryo. Higher percentages of glycosylated isoforms were associated with increasing levels of total prolactin in the pituitary gland. Thus, the synthesis of prolactin and its post-translational modifications may be important factors involved in the physiologic changes occurring around the time of hatching.

Changes in levels of immunoreactive prolactin isoforms during a reproductive cycle in turkey hens.

Changes in the ratio between immunoreactive isoforms of prolactin using Western blotting and in the total prolactin content using radioimmunoassay were measured in pituitary glands from turkey hens at different physiological stages. The type of glycosylation (N- or O-linked carbohydrates) was determined using endoglycosidase digestion (N-glycosidase F, O-glycosidase, and neuraminidase). Low levels of prolactin were observed in pituitary glands from sexually immature, out-of-lay, and molting hens. Higher levels were present during the egg-laying period and the highest levels were detected in hens which expressed incubation behavior. Two immunoreactive bands of apparent molecular weights of 24 and 27 kDa were visualized on Western blots, corresponding to the nonglycosylated and glycosylated forms of prolactin, respectively. In pituitary glands from incubating turkey hens, about 70% of the prolactin was glycosylated (27-kDa isoforms), whereas about 60% was glycosylated in immature and in hens during the first egg-laying period. In pituitaries from out-of-lay and molting hens the percentage of glycosylated prolactin was 38 and 33%, respectively. Thus, higher percentages of glycosylated isoforms (27 kDa) were associated with high levels of total prolactin and lower percentages were associated with low levels of prolactin content in the pituitary gland. Digestion of the isoforms with N-glycosidase F resulted in a single band with an apparent molecular weight of 24 kDa. Partial deglycosylation was achieved using neuraminidase, whereas digestion with O-glycosidase had no apparent effect on the isoforms. Thus it appears that the glycosylated isoforms of prolactin have N-linked carbohydrates containing sialic acid.