John D. Kirby

Sperm Mobility: Phenotype in Roosters (Gallus domesticus) Determined by Mitochondrial Function

Abstract Previously, inheritance of sperm mobility entailed a maternal additive genetic effect, and sperm ATP content was correlated (r = 0.80) with phenotype. The present study was conducted to determine if mitochondrial function was critical to phenotypic expression. Whereas phenotype was independent of mitochondrial helix length, phenotype was correlated with sperm oxygen consumption (r = 0.83) using random-bred roosters. Aberrant mitochondria characterized immobile sperm, as evidenced by transmission-electron microscopy. Such mitochondria were swollen and contained disorganized cristae. Additional experiments were performed with roosters from lines selected for low or high sperm mobility. A threefold difference in sperm oxygen consumption was observed between lines. Single nucleotide polymorphisms were observed in mitochondrial DNA by sequencing replicate mitochondrial genomes from each line. An A-to-G substitution in the gene encoding tRNAArg was inherited consistently, as evidenced by restriction fragment length polymorphism analysis using two male and two female progeny per family group and 14 family groups per line. Motile concentration in semen from low-line males was half that observed in semen from high-line males, as evidenced by computer-assisted sperm motion analysis. Likewise, 47% of sperm from low-line males contained aberrant mitochondria, compared to 4% for high-line males. In summary, sperm mobility phenotype was dependent on mitochondrial function, which in turn was altered by genetic selection. Fowl deferent duct fluid contains a high concentration of glutamate. We propose that variation in sperm mobility phenotype stems from the extent to which glutamate induces excessive mitochondrial Ca2+ uptake before ejaculation.

Reproduction in Male Birds

Reproduction is a process that can be organized into distinct developmental and functional phases. In the case of the male, these include fertilization, formation of a patent reproductive tract, production of sperm, manifestation of male-specific behavioral patterns, and expulsion of sperm from the body. This perspective can provide insight that may be missed if reproduction is viewed primarily as an isolated act. For example, although the reproductive tract is fully functional only in the adult, it is formed, for the most part, prior to hatching. Furthermore, although spermatogenesis is associated with puberty, spermatogenesis is not constrained by chronological age but rather by the extent to which testicular cells proliferate and differentiate, which in turn is coupled to the developmental limitations of gonadotropin secretion. Finally, androgens essential to the function of the reproductive tract, the appearance of secondary sexual attributes, and male behavior may have detrimental effects upon the development of immune and connective tissue if the hormonal signal appears during the period of rapid prepubertal growth and differentiation. Thus, this chapter will discuss the process of reproductive system development and function in the male bird.

Molecular cloning and functional characterization of a vasotocin receptor subtype expressed in the pituitary gland of the domestic chicken (Gallus domesticus): avian homolog of the mammalian V1b-vasopressin receptor.

The neurohypophysial hormone arginine vasotocin (AVT) stimulates adrenocorticotropin hormone (ACTH) secretion from the avian anterior pituitary gland resulting in increased adrenal secretion of corticosterone in response to stress. Here, we report molecular cloning and functional characterization of a gene encoding an AVT receptor subtype, designated the VT2 receptor, that may mediate the stimulatory effect of AVT on ACTH secretion in birds. The open reading frame predicts a 425 amino acid polypeptide that includes seven segments of 19 to 24 hydrophobic amino acids, typical of guanine nucleotide-protein coupled receptors. Phylogenetic analysis revealed that the VT2 receptor shares highest identity with the mammalian V1b-vasopressin receptor subtype. Expressed VT2 receptors in COS7 cells mediate AVT-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the domestic chicken, expression of VT2 receptor gene transcripts is limited to the pituitary gland. Based on similarities in sequence, site of expression and coupled signal transduction pathways, we conclude that the VT2 receptor is the avian homolog of the mammalian V1b-vasopressin receptor, and therefore may play an important role in the avian stress response.

Episodic Gonadotropin Secretion in the Mature Fowl: Serial Blood Sampling from Unrestrained Male Broiler Breeders (Gallus domesticus)

Abstract Forty-week-old male broiler breeders were used in two experiments. Males were reared as recommended by the breeder, housed in individual cages, and cannulated to facilitate blood sampling. In experiment 1, blood samples were collected at 10- min intervals for 4 h commencing the day of cannulation (Day 0) and for 12 h on each of Days 1 and 2. In experiment 2, blood samples were collected at 10-min intervals for 8 h on Day 1. After centrifugation, plasma was stored at −20°C until LH, FSH (experiment 1 and 2), testosterone, and corticosterone (experiment 1) concentrations were determined by RIA. Different statistical methods used to identify hormone secretion profiles revealed a characteristic pulsatile pattern of LH and FSH in plasma. However, LH pulses were more frequent and had greater amplitude than FSH pulses. Less than 32% of the FSH pulses were associated with LH episodes. Conversely, the association between LH and testosterone pulses averaged 83% in birds with testis weight greater than 10 g. Concentrations of corticosterone tended to increase after cannulation and remained elevated for only 3–4 h. Our data indicate that LH, FSH, and testosterone secretion is pulsatile in male broiler breeders. Additionally, LH pulses are associated with testosterone episodes but not with FSH pulses. The pulsatile pattern of FSH secretion, which is unique from those of LH, in adult males suggests that FSH secretion is independently regulated in the adult male fowl.

Field testing the influence of sperm competition based on sperm mobility in breeder turkey toms

1. Commercial reproduction of turkeys relies on pooling of semen from multiple males for inseminations. Understanding how sperm characteristics influence paternity under commercial breeding conditions is important to improving production efficiency. 2. The objective of this study was to evaluate progeny production of individual toms following commercial practices of pooling semen to determine if sperm mobility influences progeny production in field conditions. 3. A total of 104 toms were evaluated for sperm mobility. A subset of 10 toms were housed together and semen was collected, pooled and used to inseminate hens (n = 28). Hens were inseminated at 30 weeks of age and weekly thereafter. 4. Ejaculates from each tom were evaluated on two separate days for sperm mobility. Semen from each tom was diluted and layered upon 6% (wt/vol) Accudenz® solution. The sperm suspension was incubated at 41°C for 5 min and absorbance was measured with a spectrophotometer. 5. Toms were ranked by absorbance and categorised as high or low if mobility score was ±1 SD from the flock mean (average). 6. For parentage determination, DNA was extracted from tom, hen and poult blood. Poult parentage (n = 276) was determined at one day of age or at 14 weeks by analysis of marker genotypes that were generated by polymerase chain reaction (PCR) amplification of genomic DNA with selected microsatellite markers. 7. Sperm mobility differed across males with absorbance values ranging from 0·147 to 0·366. 8. Findings demonstrate differences in poult production among individual toms when semen from multiple males was pooled and inseminated. Toms classified as high, average and low produced 55, 41 and 4% of the offspring, respectively. 9. It appears that sperm mobility is a trait that influences sperm competition among toms under field conditions where sperm numbers inseminated from individual toms are not controlled or constant and that toms with low sperm mobility produce few offspring.

Circadian rhythm of the preovulatory surge of luteinizing hormone and its relationships to rhythms of body temperature and locomotor activity in turkey hens.

Abstract Simultaneous measurements of plasma LH, body temperature, and locomotor activity were made in laying turkey hens and are reported. Blood samples were remotely collected using a jugular cannula system, and body temperature and locomotor activity were remotely monitored using a radiotelemetry system in freely moving laying turkeys. Under a photoschedule of 14L:10D, the period for preovulatory surges of LH was 25.7 ± 0.4 h while the periods for peak body temperature and onset of sustained locomotor activity were 24.9 ± 0.4 and 25.7 ± 0.5 h, respectively. During exposure to constant light, the periods for preovulatory surges of LH, peak body temperature, and onset of sustained locomotor activity increased to 27.9 ± 0.9, 26.7 ± 0.7, and 27.4 ± 0.7 h, respectively. With the 14L:10D photoschedule, initiation of LH surges was restricted to the scotophase, but after 8 days of constant light, initiation of LH surges had dispersed throughout the 24-h subjective day and night. With constant light, the amplitude of the peak body temperature rhythm decreased, while the duration of the locomotor activity rhythm became broadened and, in some birds, disorganized. Peak body temperature and onset of locomotor activity rhythms and LH surges did not coincide, even though peak body temperature, onset of locomotor activity, and LH surges had similar periods. It is concluded that 1) the photoschedule influences the periods of the LH surge, peak body temperature, and onset of locomotor activity; and 2) a specific or direct relationship between the rhythms of LH surge, body temperature, and locomotor activity remains to be determined in laying turkey hens.

Changes in Plasma Concentrations of Luteinizing Hormone, Progesterone, and Estradiol-17β in Peripubertal Turkey Hens under Constant or Diurnal Lighting

Abstract Possible circadian fluctuations and long-term changes in concentrations of reproductive hormones in peripubertal female birds is poorly documented in comparison with mammalian species. Our objective was to document changes in concentrations of several reproductive hormones the several days before and after initial pubertal preovulatory surges of LH in turkey hens photostimulated with either constant (24L:0D) or diurnal (14L:10D) lighting. The hens were cannulated for hourly blood sampling, starting 10 days after photostimulation and continuing until all hens had laid at least two eggs. First eggs were oviposited between 16 and 24 days after photostimulation, and egg production ranged from two to nine eggs/hen during the experimental period. With both lighting treatments, concentrations of LH declined slightly, concentrations of progesterone (P4) increased, and concentrations of estradiol-17β (E2) were constant the 3–4 days prior to initial LH surges with no circadian fluctuations in hormone concentrations. Most (10 of 13) initial preovulatory surges of LH were coupled with ovulations, and all LH surges were coupled with P4 surges. Those LH and P4 surges not coupled with ovulations (blind surges) occurred with both lighting treatments, but the incidence of blind surges was higher with diurnal lighting. The interval between LH and P4 surges was longer between the first and second surges than between subsequent surges, when the interval was approximately 26 h. The duration of LH surges (7.4 ± 3.0 h) was shorter than that of P4 surges (10.0 ± 2.0 h). We conclude that, in the peripubertal female turkey, 1) prior to puberty (first LH-P4 surges), there are no circadian fluctuations in concentrations of LH, P4, and E2, 2) 3 days prior to initial LH surges, E2 concentrations are stable, LH concentrations decline slightly, and P4 concentrations increase, and 3) surges of LH are coupled to surges of P4 but LH-P4 surges are not always coupled to ovipositions (blind surges), possibly because of internal ovulations.

Heritable Sperm Degeneration in the Domestic Fowl

The domestic fowl is among the most fecund of all terrestrial vertebrates. This tremendous capacity is suggested by the fact that a single female may produce as many as 300 progeny in a single year. This ability is due to several factors, including oviparity, selection for uninterrupted egg production; and artificial incubation. Oviparity has facilitated the production of numerous progeny by allowing females to produce up to one egg per day in the absence of pregnancy and its concomitant pause in egg production.

Frequency of luteinizing hormone surges at initiation of egg laying in turkeys photostimulated with either continuous (24L:0D) or 14L:10D lighting treatments

The secretory pattern of luteinizing hormone (LH) changes after photostimulation of photosensitive turkey hens. Prior to photostimulation, LH secretion is pulsatile, with pulses of LH of short duration and relatively high magnitude imposed on a low baseline (<1 ng/ml) concentration of LH between the pulses (Chapman et al., 1994). The baseline concentration of LH increases during the first dark period following the first long-day photostimulation (Bacon and Long, 1995). This photoperiod-induced increase in the baseline concentration of LH (2–4 ng/ml) is then maintained through initiation of egg laying, when ovulatory surges of LH commence (Chapman et al., 1994). The increased baseline concentration of LH may be a key regulatory event of initial oviduct and ovarian growth as well as in increased oestrogen secretion, while the preovulatory LH surge is a key regulatory event of the hen’s ovulatory cycle and progesterone secretion. Up to this point, little has been uncovered concerning assumed changes in the pattern(s) of LH secretion associated with initiation of egg laying in turkeys. Therefore, changes in circulating concentrations of LH at around the initiation of egg laying were determined in turkeys and are reported here. Turkey hens of a line selected for increased egg production for 38 generations (Nestor et al., 1996) were given short-day (6L:18D) lighting from 16 weeks of age. At 27 weeks of age, the hens were housed in small individual hen enclosures bedded with wood shavings, and cannulated via the jugular vein (Chapman et al., 1994). The hens were returned to their individual enclosures and the cannulas connected to a swivel and tether system so the hens had freedom of movement within their individual enclosures. Feed and water were available ad libitum. The Institutional Animal Care and Use Committee approved all procedures. The hens were then photostimulated with either 24L:0D (n=8) or 14L:10D (n=8) photoperiods at 50 lux intensity. After 10 d of photostimulation, the hens were serially bled each hour for 320 h (13+ days). All of the 24L:0D birds and 6 of the 14L:10D birds completed the trial, and all hens completing the trial laid eggs (range 2–9 eggs/hen). First ovulatory surges of LH occurred 3–10 d after starting serial bleeding. Prior to first ovulatory surges of LH, the concentration of LH was relatively constant (range 2–4 ng/ml) and did not show diurnal variation under either the 24L:0D or the 14L:10D lighting treatment. First ovulatory surges of LH were relatively robust and usually resulted in laying of first eggs (11 LH surges resulted in first eggs while 3 LH surges did not). The elapsed time interval between LH ovulatory surges 1 and 2 (31·6 h) was longer than between subsequent LH ovulatory surges (26·2 h) for both lighting treatments. The interval between all subsequent LH surges (maximum n = 9) remained constant. There was no lighting treatment effect on interval length between LH ovulatory surges. In the 24L:0D lighting treatment group, a total of 54 ovulatory surges of LH were observed, but only 50 eggs were associated with these 54 ovulatory surges of LH. Thus, 4 of the 54 LH surges (7%) were not associated with eggs being laid. In the 14L:10D lighting treatment group, a total of 33 ovulatory surges of LH were observed, but only 22 eggs were associated with these 33 ovulatory surges of LH. Thus, 11 of the 33 LH surges (33%) were not associated with eggs being laid. The surges of LH not associated with laying of eggs were of the same approximate amplitude as surges of LH associated with laying of eggs. The association of progesterone surges with LH surges has been determined in 4 of the 14 hens, 2 from each lighting treatment. In these hens, 24 LH surges were observed, and these 24 surges led to the laying of 19 eggs. All 24 surges of LH were associated with surges of progesterone, indicating all LH surges occurred when an ovarian follicle capable of responding by progesterone secretion was present. Surges of LH and progesterone not coupled with egg laying may represent internal ovulations, possibly due to an inability of the oviduct to sequester the ovulated ovum. In summary, changes in secretion of LH at first ovulations in turkey hens were determined. Prior to first ovulations, a relatively high baseline concentration of LH was present, but no diurnal fluctuations were detected. Most initial ovulatory surges of LH (11/14) were associated with subsequently laid eggs. The interval between ovulatory surges was not affected by lighting treatment (14L:10D vs. 24L:0D). The interval between the first and second ovulatory surges