A. J. Davis

Expression Pattern of Messenger Ribonucleic Acid for Follistatin and the Inhibin/Activin Subunits during Follicular and Testicular Development in Gallus domesticus

The expression of mRNA for follistatin and the inhibin/activin subunits was investigated in the follicles of laying hens and the testes of immature and mature roosters. Total RNA was isolated from immature and mature testes and from individual granulosa layers of the F1 through F5 follicles, from a pool of the F6-F8 follicles, from the small yellow follicles, and from the combined granulosa and theca layers of the large white follicles. Northern blot analysis was performed, and a follistatin mRNA transcript of approximately 2. 4 kilobases (kb) was detected in immature testes and in the following follicles: F5, the pool of F6-F8, and small yellow and large white follicles. The greatest expression of follistatin occurred in the small yellow follicles, and in this tissue two minor transcripts of approximately 1.7 and 3.7 kb were also detected. The inhibin alpha subunit was expressed in both testes samples and in all of the follicles except the large white follicles. Expression of the alpha subunit was greatest in the F5 follicle, and expression decreased with follicle maturity. As previously reported, the inhibin/activin ssA subunit was found in the greatest abundance in the F1 follicle, with lesser amounts detected in the other hierarchical follicles and immature testes. In contrast to the ssA subunit, the inhibin/activin ssB subunit was not detected in the four largest hierarchical follicles but was expressed in greatest abundance in the pool of F6-F8 and small yellow follicles. This represents the first report, to our knowledge, of the detection of follistatin mRNA in the hen ovary and rooster testes. The restriction of follistatin mRNA expression to the small follicles suggests that follistatin, by regulating activin and/or inhibin availability, may play a critical role in early follicular development.

Activin A and Gonadotropin Regulation of Follicle-Stimulating Hormone and Luteinizing Hormone Receptor Messenger RNA in Avian Granulosa Cells1

Abstract Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin α subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F1 and F3 + F4 follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin α subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F3 + F4 granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F3 + F4 cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F1 and F3 + F4 granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F3 + F4 granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.

The Influence of a Twice-a-Day Feeding Regimen After Photostimulation on the Reproductive Performance of Broiler Breeder Hens

Broiler breeder hens are typically provided a restricted amount of feed once a day. This feed is rapidly consumed; therefore, the hens fast for an extended period of time before their next feeding. In the current research, the effects on reproductive performance of implementing a twice-a-day vs. a once-a-day feeding program after photostimulation were investigated. Pullets and cockerels were reared on a skip-a-day feeding program. Pullets were weighed at 20 wk of age and then distributed into 30 laying pens such that each pen had a similar BW distribution. Each individual laying pen consisted of 35 hens and 4 roosters. At 21 wk of age, the birds were photostimulated for reproduction and 15 of the laying pens were placed on a once-a-day feeding schedule, whereas the other 15 pens were placed on a twice-a-day feeding schedule. The total amount of feed provided per day to all the laying pens was the same. Birds fed once a day received all their feed at 0630 h, whereas birds fed twice a day received 60% of their total feed allotment at 0630 h and the other 40% at 1500 h. Even though both treatment groups began egg production at the end of wk 23, birds fed twice a day laid more (P < or = 0.05) eggs through 42 wk of age than those fed once a day. Additionally, the average egg weight for the entire production period, which lasted until the birds were 60 wk of age, was greater for hens fed twice a day. Overall BW uniformity for the entire experiment was significantly better for hens fed twice a day vs. once a day. However, cumulative mortality was significantly higher for hens fed twice a day than for those fed once a day. The results indicate that feeding broiler breeder hens twice a day after photostimulation may enhance reproductive performance during the early lay period.

Follicle-Stimulating Hormone Regulation of Inhibin α- and βB-Subunit and Follistatin Messenger Ribonucleic Acid in Cultured Avian Granulosa Cells

Abstract FSH regulation of inhibin α-, βB-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F4 + F5 follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P < 0.05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin α-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin α-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P < 0.05) mRNA levels for the inhibin α-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F4 + F5 granulosa cells. The effects of FSH on follistatin and βB-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin α-subunit in small prehierarchical follicles.

Impact of Feeding Program After Light Stimulation Through Early Lay on the Reproductive Performance of Broiler Breeder Hens

Skip-a-day feed restriction is a common industry management technique that is used in rearing broiler breeder pullets. Often pullets are maintained on a skip-a-day feeding program after they have been photostimulated for reproduction, and in some cases until 5% egg production is reached. The current research examined whether providing daily nutrient intake during the critical period of ovarian development that follows photostimulation for reproduction improves subsequent egg production. Pullets and cockerels were fed on a skip-a-day basis during rearing. Pullets were weighed at 20 wk of age and then distributed into 30 laying pens such that each pen had a similar BW distribution. Each individual laying pen consisted of 35 hens and 4 roosters. At 21 wk of age, the birds were photostimulated for reproduction; 15 of the laying pens were placed on an every-day feeding schedule, whereas the other 15 pens were maintained on a skip-a-day feeding schedule until they reached 8% egg production at 26.5 wk of age. From 26.5 to 65 wk of age, all hens were fed on an every-day basis. The CV of BW did not differ between the hens of the 2 treatment groups at any point from 21 to 64 wk of age. Weekly percentage hen-day egg production was greater (P < 0.05) in the hens fed on the every-day versus skip-a-day program after photostimulation from wk 25 of age to 65 wk of age except for wk 29, 30, 35, 37, 39, 46, and 47 of age. Total hen-day egg production through 65 wk of age in the hens that were provided feed every day after photostimulation was greater (172 vs. 155 eggs/hen) than in hens fed on a skip-a-day basis until 26.5 wk of age. These results suggest that continuing skip-a-day feeding after photostimulation until reaching 8% egg production does not improve BW uniformity, but does cause lasting reproductive dysfunction in broiler breeder hens.

Molecular Cloning and Expression Analysis of the Complementary Deoxyribonucleic Acid for Chicken Inhibin/Activin βB Subunit1

Abstract Inhibins and activins are dimeric peptide hormones that play an integral role in the intraovarian regulation of folliculogenesis. The domestic hen, with its well-defined follicular hierarchy, provides a unique model in which to study the role of these hormones in follicular development. In the present study, the complete coding sequence and deduced amino acid sequence for the chicken inhibin/activin βB subunit has been determined from cDNA clones isolated from a chicken ovarian granulosa cell library. This βB-subunit cDNA predicts a precursor protein of 392 amino acids containing the mature C-terminal 115 amino acid βB subunit. When compared to the βB subunit isolated from a variety of species, the chicken cDNA clone showed high nucleotide identity in the full-length coding region (>70%) and in the mature coding region (>80%). In addition, the deduced amino acid sequence of chicken βB subunit showed greater than 95% identity compared to other species in the mature peptide region. Expression of the βB-subunit mRNA was detected by reverse transcription-polymerase chain reaction in both gonadal and extragonadal tissues. Northern blot analysis detected expression in the gonadal tissues only, specifically in the granulosa tissue from the F3-F5 follicles, small yellow follicles (SYF), large white follicles, and immature and mature rooster testes. A major transcript of approximately 4.1 kilobases (kb) and three minor transcripts of approximately 8.4 kb, 6.5 kb, and 1.7 kb were detected in the SYF granulosa samples. To examine the expression pattern of the βB subunit around the stage of follicle selection, the SYF granulosa was subdivided into two groups: 6–8 mm and 9–12 mm. Quantification of RNA expression (n = 3) showed that expression of the βB subunit was maximal in the 6–8 mm SYF. Activin B, as well as other intraovarian signals, may regulate early follicle selection and/or development in the chicken.

The mRNA for zona pellucida proteins B1, C and D in two genetic lines of turkey hens that differ in fertility

The avian inner perivitelline layer (IPVL) contains zona pellucida protein-B1 (ZPB1), zona pellucida protein-C (ZPC) and zona pellucida protein-D (ZPD). These three proteins may be involved in sperm binding to the IPVL. ZPB1 is produced by the liver and transported to the developing preovulatory follicle, while ZPC and ZPD are synthesized and secreted by the granulosa cells of the preovulatory follicle. The mRNA of ZPB1, ZPC, and ZPD was investigated in two lines of turkey hens selected for over 40 generations for either increased egg production (E line) or increased body weight (F line). Total RNA was extracted from the liver and from 1cm(2) sections of the granulosa layer around the germinal disc and a nongerminal disc area of the F(1) and F(2) follicles of hens from each genetic line. Northern analysis was performed using chicken cDNA probes for all three ZP proteins. Hepatic mRNA for ZPB1 was greater (P<0.05) in turkey hens from the E line than the F line. Although, there was no difference in ZPC mRNA between the germinal disc and nongerminal disc region of the two largest follicles in E line hens, ZPC mRNA was greater in the nongerminal disc region compared to the germinal disc region in the two largest follicles obtained from the F line hens. There were no differences in ZPD mRNA between the germinal disc and nongerminal disc regions of the F(1) and F(2) follicles for either genetic line. The results suggest that the greater rates of fertility previously observed in eggs from the E line hens compared with the F line of hens may be related to differential amounts of the potential sperm binding proteins ZPB1 and ZPC.

Impact of Extended Heat Treatment on the Amino Acid Digestibility and TMEn Content of a Formaldehyde-Treated Diet

SUMMARY Eliminating the contamination of broiler carcasses by pathogens such as Salmonella that can cause human illness continues to be a tremendous challenge and concern for the poultry industry and government regulatory agencies. One source for the introduction of these pathogens in poultry production can be the feed. Extended heat conditioning of diets prior to pelleting combined with the addition of preservatives such as formaldehyde has proven effective in reducing the risk of Salmonella in poultry diets. However, there has been a concern that the addition of some forms of formaldehyde to diets subjected to extended heat treatment could decrease the metabolizable energy and amino acid availability of the diet. To partially address this question, the TMEn and digestible amino acid coefficients of a broiler starter diet treated with 0 (control) or 0.3% formaldehyde solution prior to heat treatment at 82°C for 4.5 min were determined. Cecectomized and intact adult Single Comb White Leghorn roosters were used for determining amino acid digestibility and TMEn, respectively. The addition of the formaldehyde compound did not affect either the TMEn or the digestible amino acid content of the diet, except for a marginal (less than 1%) decrease in arginine digestibility. These results indicate that the supplementation of poultry diets with formaldehyde solution prior to extended heat treatment to eliminate Salmonella contamination did not impact the nutritional value of these diets for poultry.

Zona pellucida protein B2 messenger ribonucleic acid expression varies with follicular development and granulosa cell location1

ABSTRACT The freshly ovulated ovum in avian species is surrounded by a protein layer called the inner perivitelline layer (IPVL). The IPVL contains zona pellucida proteins and 6 distinct zona pellucida genes have been identified (ZPA, ZPB1, ZPB2, ZPC, ZPD and ZPX1) in the chicken. In the present research, the expression of the mRNA for ZPA, ZPB2, and ZPX1 was investigated in 2 lines of turkey hens selected for either increased egg production (E line) or increased body weight (F line). Theca and granulosa cell expression of the mRNA for ZPA and ZPB2 was also investigated in hierarchical and prehierarchical follicles from broiler breeder hens. Granulosa tissue was collected from F1 through F4 and F1 through F10 follicles in E line and F line hens, respectively. A one cm2 section of the granulosa layer around the germinal disc (GD) and an equivalent sized nongerminal disc (NGD) area was also collected from the F1 and F2 follicles from other hens from each genetic line. Granulosa and theca tissue was collected from hierarchical and prehierarchical follicles of broiler breeder hens. Total RNA was extracted from the samples. Minor groove‐binding probes and primers for detecting ZPA, ZPB2, and ZPX1, were made for real‐time PCR analyses. Expression of ZPA, ZPB2, and ZPX1 was detected in all follicle sizes from both genetic lines of hens. No significant differences in ZPA and ZPX1 mRNA expression were detected between the GD and NGD granulosa cells. However, the expression of the mRNA for ZPB2 was significantly greater in the GD granulosa cells when compared to the NGD granulosa cells in F1 and F2 follicles from E line and F line hens. In broiler breeder hens, the mRNA expression of ZPA and ZPB2 was greatest in the smallest prehierarchical follicles. The results suggest that higher expression of ZPB2 in the germinal disc area may be important for the preferential binding of sperm to this region of the IPVL.

Foot ash can replace tibia ash as a quantification method for bone mineralization in broilers at 21 and 42 days of age

&NA; Prior research indicated that foot ash determinations were as robust as tibia bone ash determinations in reflecting the degree of bone mineralization in chicks at 14 d of age. In the current research, the relative effectiveness of the 2 procedures was evaluated in 21‐ and 42‐day‐old broilers while also evaluating a new dietary phytase supplement. In experiment 1, broilers were fed until 21 d of age a negative control diet with 0.24% available phosphorus, a positive control diet with 0.48% available phosphorus, or the negative control diet supplemented with 300, 500, 1,000, or 2,500 phytase units/kg diet. In experiment 2, broilers were fed until 42 d of age negative control diets having 0.275, 0.250, and 0.225 percent available dietary phosphorus in the starter, grower, and finisher periods, respectively, positive control diets having 0.475, 0.450, and 0.425 percent available dietary phosphorus in the starter, grower, and finisher periods, respectively, or the negative control diets supplemented with 500, 1,000, or 2,500 phytase units/kg diet. At 21 and 42 d of age, broilers fed diets supplemented with the 2 highest doses of phytase had foot and tibia ash values equal to those fed the positive control diet and higher than those fed the negative control diet. At 42 d of age, feed conversion and total breast meat yield values for the broilers fed the highest dose of phytase were superior to the values of the birds fed the positive control diet or the diet containing the lowest dose of phytase. The results indicate that adding levels of this new dietary phytase beyond what is necessary for normal bone mineralization enhances feed conversion and that dried foot and tibia bone ash determinations are both reliable in detecting differences in bone mineralization in 21‐ and 42‐day‐old broilers.