Physiology and Reproduction

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Group 1, 5 treatments. (trts): 1) ... trt at the same age as Group 1 trt 3 and trt 5, respectively. Birds in ... Sperm-associated antigen 6, a flagellar protein important.

Physiology and Reproduction 86 Effect of early-life nutritional stress on the expression of vasotocin and corticotropin-releasing hormone receptors in liver and anterior pituitary of male broilers. Seong Kang* and Wayne Kuenzel, Department of Poultry Science, University of Arkansas, Fayetteville, AR. The transportation of post-hatched chicks induces early-life nutritional stress and influences growth performance. Mammalian vasopressin receptor 1a (V1aR), and corticotropin-releasing hormone (CRH) receptors in liver are involved in the metabolic disorder affecting glucose homeostasis. The objective of this study was to test the hypothesis that the post hatch nutritional stress (feed deprivation, FD) causes the differential changes of the avian vasotocin receptors type 1a (V1aR) and type 1b (V1bR), as well as CRH- receptor type 1 (CRH-R1) and type 2 (CRH-R2) expression in liver and anterior pituitary of broilers. Dayold chicks were randomly selected into 2 groups. Group 1, 5 treatments (trts): 1) 0hr controls (Con), 2) 12hr feed (12hF), 3) 12hFD, 4) 36hF, and 5) 36hFD. Trt 1 was sampled 0hr, trt 2 and 3 at 12hr while trt 4 and 5 at 36h. Anterior pituitaries (AP), liver and blood samples were collected for all trts (n = 14/trt). Group 2, 3 trts: 1) controls (Con2), 2) 12hFD2, and 3) 36hFD2. Group 2 trt 2 and trt 3 experienced the same trt at the same age as Group 1 trt 3 and trt 5, respectively. Birds in Con2 had full feed throughout the experiment. All birds in Group 2 were sampled at 6 weeks of age, including AP, liver and blood (n = 10–12/trt). One-way ANOVA was performed using the JMP 11 followed by mean comparison using the Tukey’s HSD test. Results showed that CORT levels increased in 12hFD and 36hFD compared with controls (12F and 36F). No significant difference of CORT was observed in between 12hFD2 and 36FD2 compared with Con2. Results of gene expression study showed that V1aR, CRH-R1 and CRH-R2 in AP were decreased by 12hFD compared with controls (P < 0.05). V1aR and CRH-R2 in AP were increased by 36hFD (P < 0.05). V1bR and CRH-R1 in AP were decreased by 36hFD (P < 0.05). In liver, V1aR was increased by 12hFD and 36hFD. There were no significant changes in V1bR, CRH-R1 and CRH-R2 expression by 12hFD and 36hFD. At 6 weeks of age, V1aR, V1bR, CRH-R1 in AP, and V1aR, CRH-R1 and CRH-R2 in liver in 36hFD2 birds were increased compared with Con2 and 12hFD2 (P < 0.05). CRH-R2 in AP, and V1bR, CRH-R1, and CRH-R2 in liver of the 12hFD2 birds were decreased compared with controls (P < 0.05). Taken together, results suggest that 12hFD and 36hFD stress in post hatch chicks cause differential effects to chicken VTR and CRHR expression in the early-life period. The upregulation of the VTRs and CRHRs in the 36hFD compared with 12hFD broilers persisted through 6 weeks of age. Key Words: nutritional stress, liver, anterior pituitary, vasotocin receptor, CRH receptor

87 Cellular evidence supporting an additional structure in the chick brain involved in the neuroendocrine regulation of the stress response. Wayne Kuenzel*1, Gurueswar Nagarajan1, Alexander Jurkevich2, and Seong Kang1, 1University of Arkansas, Fayetteville, AR, 2University of Missouri, Columbia, MO. The traditional neuroendocrine system regulating stress is the hypothalamo-pituitary-adrenal (HPA) axis. A recent study involving broilers subjected to a stressor, food deprivation, revealed that the first brain structure responding to that stressor was not in in the paraventricular nucleus (PVN) of the hypothalamus. The first response occurred in the septum, in a structure called the nucleus of the hippocampal commissure 32

(NHpC). The NHpC displayed a marked increase in mRNA that codes for the stress hormone corticotropin-releasing hormone (CRH) (Nagarajan et al., 2017, Neurosci. Lett., 642:14–19). Experiments were designed to obtain cellular evidence supporting the NHpC as a structure in the HPA axis. Brain slice culture experiments were performed using chick brain slices, sectioned at 350μm and placed in cell culture inserts supplied with a standard culture media to keep cells viable. Slices were maintained in a culture environment for 9–10 d set at 36C with 95%O2, 5.0% CO2 before the initiation of treatments. Within the NHpC an arginine vasotocin (AVT) terminal field was previously found along with CRH neurons and receptors for AVT, vasotocin 1a receptors (V1aR). Therefore AVT was administered to slices containing the NHpC. Additionally a specific blocker of the avian V1aR known as SR-49059 was also applied to slice cultures. The purpose was to determine whether AVT would activate CRH neurons and the antagonist, SR-49059, would attenuate CRH gene expression. Three treatment (trt) groups: AVT administration, AVT + SR-49059, and controls (n = 6/trt) were formed. An ANOVA followed by a mean separation test, the lsd, were performed. Indeed AVT significantly increased CRH mRNA (P ≤ 0.05). When the blocker was applied before AVT administration, an increase in CRH gene expression was not observed. Curiously, the avian V1aRs were not found on CRH neurons. Immunocytochemistry revealed that V1aRs were abundant in glia within the NHpC. Results strongly suggest a possible intercellular communication between glia and CRH neurons. One candidate serving that role is brain derived neurotrophic factor (BDNF), since BDNF was activated by AVT administration. Additional data showed that the AVT response of BDNF was not observed when SR49059 was applied to brain sections containing the NHpC. It is unknown whether the source of BDNF released by AVT trt was from glia and/or neurons within the NHpC. Supported in part by an Arkansas Biosciences Institute Grant. Key Words: broiler, septal brain region, corticotropin-releasing hormone, BDNF 88 Sperm-associated antigen 6, a flagellar protein important in mammalian fertility, is sequentially expressed in the male reproductive tract and associated with increased sperm mobility in the rooster. Andrew Benson and Muslah Ahammad*, University of Georgia, Athens, GA. Sperm mobility has been shown to be a primary determinant of fertility in domestic fowl. To initiate fertilization, avian sperm cells rely on the propulsive forces generated by their flagella to reach sperm storage tubules and the site of fertilization in the oviduct as well as penetrate the investments of the egg. In mice, sperm-associated antigen 6 (SPAG6) is a scaffold protein that maintains the structural integrity of the sperm flagella and is essential for sperm mobility and fertility. Due to its known role in mammals and the significance of mobility in avian reproductive fitness, the expression of SPAG6 was investigated in the domestic chicken. In the present study, we examined the male reproductive tissues and sperm with differential mobility for the expression of SPAG6. Percoll density gradient centrifugation and swim-up migration sedimentation technique were used to separate high, medium and low mobile sperm. The expression of SPAG6 from sperm lysates isolated from the testes, epididymides, vas deferens, and ejaculated sperm of different mobility were subjected to quantitative western analysis. Quantitative Western blot analysis determined the intensity of SPAG6 expression was significantly increased at different stages of sperm maturity from testes through ejaculation. The fold difference values for testicular, epididymal, Poult. Sci. 96(E-Suppl. 1)

vas deferens and ejaculated sperm were 1.00 ± 0.00, 1.35 ± 0.03, 2.11 ± 0.16 and 2.32 ± 0.30, respectively; P < 0.05. Expression of SPAG6 was also greatest in highly mobile sperm. The fold difference values for sperm with high, medium and low mobility were 1.00 ± 0.00, 0.78 ± 0.06 and 0.72 ± 0.05, respectively; P < 0.05. The Accudenz assay was used to group roosters based on consistently producing sperm of low and high mobility. The expression of SPAG6 was significantly greater in sperm from roosters of the high mobility group (1.00 ± 0.00) followed by the low group (0.87 ± 0.03). Immunocytochemical analysis confirmed the localization of SPAG6 in the axoneme of the mid and principal piece of the sperm flagellum. The findings of the present study demonstrate that SPAG6 is localized in the axoneme of the rooster sperm flagellum, is sequentially expressed in the male reproductive tract and that its expression is directly correlated with sperm mobility. It is suggested that the SPAG6 might be used as an easily accessible marker for selection of broiler breeders with high-mobility or fertility potential. Key Words: SPAG6, sperm, immunocytochemistry, mobility, Western blot

89 The effect of incubation temperature and time of hatch on muscle growth and morphology. Daniel Clark*1, Kim Walter2, and Sandra Velleman1, 1The Ohio State University, Wooster, OH, 2Aviagen, Inc., Albertville, AL. The adult myogenic population of stem cells, called satellite cells, initially develop and become active in late-term embryos. Satellite cells are the only cell with the capacity to repair damaged myofibers and increase posthatch growth. Altering satellite cell activity may effect posthatch growth and muscle morphology. The objective of the current study was to determine if elevated incubation temperatures and time of hatch impact growth and pectoralis major (p. major) muscle morphology. Eggs were incubated at a constant 37.8°C; however, from d 14 to 18 the eggs were subject to 39.5°C for either 0, 3, or 12 h per day. Chicks were divided into early, mid, or late hatch groups based upon the time they emerged from the shell. Feed efficiency and body weight were evaluated throughout the trial while meat quality and muscle morphology was evaluated at the end of the 63 d trial. The chicks incubated at an increased temperature for 12 h per d had reduced (P < 0.01) body weights from 5 to 63 d of age compared with the 3 h treatment and control. There were no differences in body weight between the early, mid, or late hatch groups at any age, except at the time of processing when the early hatch group had an increased (P < 0.01) body weight compared with mid and late hatch broilers. Chicks from the 12 h incubation treatment had an increased (P = 0.01) gain to feed ratio compared with the control. Broilers from the 12 h incubation treatment had lower (P < 0.01) body and p. major weights compared with the 0 and 3 h treatments. Early hatch broilers had heavier body and p. major weights (P < 0.01) compared with mid and late hatch groups. The 12 h incubation treatment also reduced the number of broilers with moderate to severe fibrotic and necrotic attributes associated with myopathic p. major muscle compared with the control. Similarly, there were fewer late hatch birds with fibrotic and necrotic p. major muscles compared with the early hatch group. Ultimate pH was increased (P = 0.02) in early hatch broilers compared with the mid and late hatch groups; whereas, incubation temperatures did not affect (P = 0.03) ultimate pH. Together, these data demonstrate that altering incubation temperature is a feasible management strategy to improve muscle morphology without negatively affecting meat quality parameters. Key Words: broiler, chicken, incubation temperature, time of hatch, muscle morphology Poult. Sci. 96(E-Suppl. 1)

90 Sentinel cells of ducks complicate interpretation of the H/L ratio. Paul Cotter*, Cotter Laboratory, Arlington, MA. The purpose is to establish blood pictures (hemograms) of ducks by a combination of gross and microscopic study. Blood from 12 commercially housed ducks (6 drakes and 6 hens) at 22 wk is the data source. Sedimentation rates and hemoglobin scores composed the gross data, and standard differential counts (SDC) of Wright’s stained blood composed the microscopic data. Total white blood cell counts (TWBC) were estimated by extrapolation from the SDC slides. Linear regression of sedimentation rates determined by 5 measurements gave an estimate of −1.2 mm/hr (R2 = 0.95). Hemoglobin scores, estimating erythrocyte fragility, were based on a subjective scale ranging from 0 – 3. The average of 0.67 (+/− 0.2) did not differ between sexes (P = 0.53). In standard differential counts, leukocytes (2 × 200) were sorted as classic, variant, and typical heterophils (HC, HV, HT) small, medium, and atypical lymphocytes (Ls/Lm) monocytes, basophils, and eosinophils. Sentinel cells, including atypical heterophils, plasmacytes with segmented nuclei (Tau cells) and giant cells were commonly seen. In several samples, autophagocytosis was evident. Heterophil lymphocyte ratios (H/L 1, H/L 2) and their difference (d H/L) were computed from SDC data. H/L 1 = (HC + HV + HT)/(Ls), H/L 2 = (HC + HV + HT)/ (Ls + Lm) and d H/L = (H/L 1 – H/L 2). H/L ratios were higher in drakes (H/L 1 ~10, P < 0.03, H/L 2 ~5.5, P < 0.01) than in hens (H/L 1 ~2, H/L 2 ~1.3). The average TWBC of 65K/μL for drakes and 59K for hens were not different (P = 0.48) but were at leukocytosis/leukemoid reaction levels. Collectively, complex hemograms characterized by a variety of atypical sentinel cells occur in commercial ducks. They indicate informative blood pictures require both gross and microscopic observations. The observations are useful in determining stress, estimating welfare, and evaluation of experimental treatments. They add to basic hematological information of an important food species. Key Words: complex hemogram, duck, hematology, Tau plasmacytes

91 Glucose and uric acid in blood of broiler chicken. Ricardo Nunes*1, Jomara Broch1, Lucas Wachholz1, Cleison Souza1, Cinthia Eyng1, Paulo Pozza2, and Gene Pesti3, 1Universidade Estadual do Oeste do Paraná–UNIOESTE, Marechal Cândido Rondon, Paraná, Brazil, 2Universidade Estadual de Maringa, Maringa, Parana, Brazil, 3University of Georgia, Athens, GA. Sodium fluoride is the main anticoagulant used for the quantification of blood glucose. There is evidence that the period of fasting may influence some blood metabolite measurements. Thus, this study was to evaluate the effects of time of fasting, the use of an anticoagulant (NaF) and storage time on the metabolites uric acid and glucose present in the blood of broilers. Sixty (60) male broilers were grown to 45 d of age with an average weight of 3264 ± 235 g. They were fasted for 1 h and then fed for 30 m. After feeding, blood was collected from 10 birds (postprandial or time zero bleeding). Subsequently blood was collected at intervals of 2 h during a further 12 h fast. The blood was collected from the ulnar vein, using vacuum tubes with and without anticoagulant (NaF). The blood remained at rest for 15 m and was centrifuged at 2500 rpm for 10 m. Plasma and serum fractions were placed into Eppendorf tubes. The fractions were refrigerated (−20°C) for 30 and 60 d. Analyses were done using a biochemical autoanalyzer (Flexor EL 200) The results were analyzed using the GLM procedure in SAS. There were no observed interactions between the fraction, storage or time period of fasting. Glucose levels were higher (P = 0.004) in the serum (248 ± 25) compared with the amount present in the plasma (234 ± 17). Serum coagulation may be inhibit the enzyme enolase, preventing the use of glucose by leukocytes and erythrocytes. The time of fasting effected glucose levels 33