Immunization of chickens with quail and turkey perivitelline membrane proteins: production of antibodies and their effects on fertility.

The cross-reactivity of antibodies developed against zona pellucida proteins and their subsequent deleterious effect on fertility in heterologous species is well documented. However, similar investigations have not been undertaken in avian species. In Experiment 1, White Leghorn hens were immunized with intact germinal discs (GD) of quail and chickens. Chicken GD proteins did not elicit an immune response in chicken hens, whereas quail GD proteins were highly immunogenic. Anti-quail GD antibodies did not bind with chicken inner perivitelline membrane (IPM) proteins as determined by immunoblot analysis. To examine the fertility of immunized hens, artificial insemination was performed at weekly intervals for 4 wk following the booster immunization. No significant differences were detected in fertility or hatchability of immunized hens when compared with unimmunized control hens. In Experiment 2, White Leghorn hens were immunized with intact turkey GD, solubilized turkey perivitelline membrane (PM) modified with dinitrophenol (DNP), and solubilized chicken IPM modified with DNP. High titers of antibodies were detected against the turkey GD and the DNP-modified turkey PM proteins. A weak immune response was observed in hens immunized with modified chicken IPM proteins. The fertility and hatchability of eggs laid by immunized hens, however, were not significantly different from those of unimmunized hens. Antibodies from immunized hens were further analyzed using an in vitro assay that assesses sperm penetration of intact IPM. Sperm penetration of intact IPM was inhibited to the same extent when IPM was preincubated with preimmune as well as anti-PM immunoglobulins. Collectively, these results suggested that the antibodies developed in these hens did not cross-react with the chicken IPM proteins involved in sperm-egg interaction and thus did not influence the fertility.

Development of a novel fluorescence technique for quantifying the total number of spermatozoa stored in the uterovaginal junction of hens.

A technique was developed to determine the total number of spermatozoa stored in the uterovaginal junction of hens. After insemination of spermatozoa treated with the nuclear fluorescent dye bisbenzimide, oviductal tissue was collected from hens and homogenized. Samples of homogenate were dried, and the number of spermatozoa mm-2 was determined with the use of a fluorescence microscope. When spermatozoa were added to excised uterovaginal junction tissue before homogenization, results indicated a 1:1 linear relationship between actual numbers of spermatozoa added to the tissue and calculated numbers of spermatozoa added to the tissue. This new technique was used to show that insemination of hens with 25, 50 or 100 x 10(6) spermatozoa resulted in a linear increase in the number of spermatozoa stored in the uterovaginal junction. Insemination of hens with 328 x 10(6) spermatozoa produced no increase in uterovaginal junction storage of spermatozoa over insemination with 100 x 10(6) spermatozoa. At the maximum sperm storage tubule filling dose of 100 x 10(6) spermatozoa, only 0.22% of the spermatozoa inseminated were found in the uterovaginal junction 24 h after insemination. Treatment of spermatozoa with bisbenzimide had no detrimental effects on fertility or penetration rates when compared with untreated (control) spermatozoa. However, when spermatozoa were treated with bisbenzimide, hatchability of fertile eggs was reduced. In conclusion, this new fluorescence technique appears to be valuable in determining the total number of spermatozoa stored in the uterovaginal junction of hens.

The male contribution to broiler breeder heat-induced infertility as determined by sperm-egg penetration and sperm storage within the hen's oviduct.

The purpose of the present study was to define the role of the male broiler breeder in heat-induced infertility. Seventy-two Arbor Acres roosters were individually caged at 21 wk of age and divided equally among three heated (H) and three control (C) temperature chambers. Control temperature chambers were held at 21 C. After an 8-wk pretreatment period (20 C), an 8-wk treatment period was conducted in which the temperature in all three of the H chambers was varied from week to week according to the following schedule: Week 1, 27 C; Week 2 through Week 4, 32 C; and Week 5 through Week 8, 21 C. On a weekly basis, semen was pooled by room and inseminated into 12 groups of 10 hens each (2 groups per room). During the 1st wk when males were maintained at 27 C for 12 h, in vivo sperm-egg penetration was reduced by 48% as compared to data obtained when males were maintained at 21 C. Fertility, in vivo sperm-egg penetration, and uterovaginal sperm storage were decreased when semen from males exposed to 32 C was used to inseminate hens as compared to insemination with semen from C males. However, during this same period, the ability of sperm to bind and penetrate the egg, as determined by in vitro sperm-egg penetration, was similar between sperm from C and H males. After lowering the temperature in the H chambers back to 21 C, in vivo sperm-egg penetration as a result to insemination with semen from H males was analogous to results obtained when C males were used for insemination. Immediately after decreasing the temperature in the H chambers, fertilization of eggs by sperm from H males increased to a level similar to that obtained when eggs were fertilized by sperm from C males but then declined again during the later weeks.

Age effect of male and female broiler breeders on sperm penetration of the perivitelline layer overlying the germinal disc.

An experiment was conducted to test the effect of age (male and female) on the number of spermatozoa penetrating the perivitelline layer (PL) overlying the germinal disc (GD) in broiler breeders. Eighty young broiler breeder hens (39 wk old, Y), and 80 old spent broiler breeder hens (69 wk old, O) were randomly divided into eight groups of 20 hens each by age. Hens were inseminated weekly for 4 consecutive wk with 5 x 10(7) pooled sperm/50 microL from either young or old broiler breeder males. Sperm penetration (SP) of the PL at the GD was assessed in a random sample of 12 oviposited eggs from each hen group for each day postinsemination, with the remainder of the eggs incubated for 10 d to obtain fertility values. For the main effect of sex, and for age within sex, there were differences in mean SP (7.3 vs 4.8; Y vs O hens; P < 0.02) and fertility (73.7 vs 54.9%; Y vs O hens; P < 0.002) values. Old males had higher mean SP values and fertility (7.2 and 70.6%) than young males (4.8 and 58.0%; P < 0.03 and 0.01, respectively). Following artificial insemination of a constant number of sperm, age of hens appears to contribute more to the decrease in SP and fertility than the age of male broiler breeders. Eggs were obtained from naturally mated broiler breeder flocks from different strains (A and B), lines (male and female), and ages. There was an effect on overall mean SP values due to strain (105.8 vs 78.6 holes per GD area; Strains A and B, respectively; P < 0.0001), and line within Strain B (106.4 vs 50.8 holes per GD; male and female line, respectively; P < 0.0001). There was a quadratic relationship between SP of the PL and age in Strain A with values ranging from 153.3 to 20.0 holes per GD area (P < 0.003). In Strain B, SP holes in the PL decreased in the male line due to age (127.8 to 59.7 per GD; P < 0.01), with an effect of age on the female line also (62.1 vs. 37.8 holes per GD; P < 0.05).

Influence of male broiler breeder dietary energy intake on reproduction and progeny growth.

A study was conducted to test the effects of dietary energy intake on reproduction in genetically similar broiler breeder males and on the subsequent growth of their progeny. Fifty-nine 1-d-old pedigree broiler breeder male chicks were raised to breeding age. At 33 wk of age, 33 males were chosen and placed in one of three groups of 11 males per group and fed either 370, 330, or 290 kcal per bird per d. Each group contained both full and half brothers and had similar 6- and 33-wk mean body weights. There was a significant negative effect of decreased dietary energy intake on sperm concentration and total live sperm per milliliter of ejaculate, whereas there was no significant effect on ejaculate volume or percentage dead sperm per ejaculate. Four groups of hens (21 wk of age) with 18 hens per group, were randomly assigned to each male dietary treatment group. Hens were artificially inseminated with 50 microL neat pooled semen from one of the three male treatment groups. There was a significant linear effect of diet on fertility, with no significant effect on hatch of fertile, hatch of eggs set, or embryonic mortality. There was no effect of sire energy intake on offspring body weights at 0, 3, or 6 wk of age. Hens were similarly artificially inseminated and sperm penetration determined for 9 consecutive d postinsemination. There was a significant quadratic relationship between sperm penetration of the perivitelline layer overlying the germinal disc and day postinsemination for each of the three male treatment groups. In addition, mean sperm penetration was 62.3, 42.9, and 6.6 holes in the germinal disc perivitelline layer for the high, medium, and low energy groups, respectively. Following 16 wk of dietary energy treatment, there was a significant linear effect of diet on mean testes weight, mean testes weight as a percentage of male body weight, and male body weight.

Quantitative determination of spermatozoa penetration of the perivitelline layer of the hen's ovum as assessed on oviposited eggs.

A technique was developed to assess the number of cock spermatozoa penetrating the perivitelline layer (PL) in oviposited eggs in vivo. Two trials were conducted to test this technique and to establish correlation values between fertility and sperm penetration (SP). First, three Athens Canadian Randombred males, previously tested as having high fertility (100%), were each housed with seven hens. Sperm penetration was determined from eggs laid over a 3-d period (n = 41) with the mean number of spermatozoa penetrating the PL overlying the germinal disc (GD; 1.35 mm2 area) and nongerminal disc (NGD) areas being 162.8 and 8.4, respectively. Following removal of the males, SP was monitored to establish its duration with an average of 4.6 eggs analyzed per male per day. Mean sperm penetration during this period declined from 167.0 to .2 and from 9.2 to 0 for the GD and NGD regions, respectively. The mean duration of SP was 15.7 and 11.3 d for the GD and NGD PL, respectively. The duration of fertility was also established to be 14.0 d. There was a positive correlation between sperm penetration of the GD PL and fertility from eggs laid by naturally mated hens (r = .89, P < .001). In the second trial, three groups (1, 2, or 3) of 16 hens (35 wk of age) each were artificially inseminated weekly for 4 consecutive wk with either 100, 50, or 25 million sperm/50 microL, respectively. Inseminations were repeated weekly for 12 consecutive wk. Mean values were obtained from each of three 4-wk periods and used as replicates. Mean SP values from the GD PL for Groups 1, 2, and 3 were 402, 19.5, and 14.1, with fertility values of 95.8, 92.4, and 83.3%, respectively. Each replicate mean was obtained from approximately 24 eggs per group per day postinsemination. A significant correlation between SP of the GD PL and fertility (r = .90, P < .001) was established using artificial insemination of hens.

Fertility of male and female broiler breeders following exposure to elevated ambient temperatures.

Because elevated ambient temperatures decrease fertility, this study was designed to segregate the male and female contribution to heat stress infertility in broiler breeders. Eighty hens and 16 roosters at 21 wk of age were divided equally among two heat stress (S) and two control (C) temperature chambers. For a 10-wk pretreatment period, all birds were maintained at an ambient temperature of 21.1 C and 40% relative humidity. Following the pretreatment period, birds in the S chambers were acclimated for 1 wk at a constant temperature of 29.4 C after which the temperature in the S chambers was increased to 32.2 C for 8 wk. The temperature in the two C chambers was maintained at 21.1 C. Hens in each chamber were artificially inseminated on a weekly basis with 5 x 10(7) sperm per 50 microL from either C or S males. Egg production, semen volume, spermatocrit, and percentage dead sperm were similar during the acclimation period, even though body temperature was significantly elevated in S birds (41.8 vs 41.3 C). Sperm penetration of the perivitelline layer overlying the germinal disc (GD) was decreased in eggs from hens inseminated with semen from S males compared to eggs from hens inseminated with semen from C males (9.5 vs 23.4 sperm per GD). Following the acclimation period, body temperature remained elevated in the S birds compared to the C birds (42.2 vs 41.3 C). Also, egg production was depressed in the S vs C hens (55.8 vs 82.9%). Semen volume, spermatocrit, and percentage dead sperm were not affected by S treatment. However, when hens were inseminated with semen from S males, sperm penetration of the perivitelline layer overlying the GD and egg fertility were decreased compared to hens inseminated with semen from C males (5.4 vs 14.9 sperm per GD, 45.5 vs 73.8% fertility). In conclusion, the male bird appears to contribute more to heat stress infertility than the female.

Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the hen's ovum.

In vitro incubation of cock spermatozoa with perivitelline layer (PL) from recently ovulated ova of the hen resulted in binding of spermatozoa to the PL and activation of the acrosome reaction. A simple quantitative technique was developed for assessing these events. Following incubation of the PL (0.5 cm2 sections) with spermatozoa, the PL section was rinsed and stained with Schiff's reagent. Microscopic examination revealed holes in the PL that were assumed to be sites of spermatozoa penetration. Utilizing this technique, a correlation was demonstrated between sperm concentration and the number of spermatozoa attaching to the PL and undergoing an acrosome reaction. Pre-treatment of spermatozoa with solubilized PL inhibited spermatozoa binding to pieces of intact PL. The PL overlying the germinal disc and a similarly sized section of PL from another area of the ovum were removed and incubated separately with spermatozoa (1 x 10(5) sperm/100 microliters). Spermatozoa showed preferential attachment and digestion of the PL from the germinal disc area (809 sperm/mm2) as compared to PL from other areas of the ovum (608 sperm/mm2). Spermatozoa attached to the PL in a circular, doughnut-shaped fashion in the area directly over the germinal disc.

Cross-reactivity of sperm-binding proteins from chicken, turkey, and duck oocytes.

Binding and penetration of spermatozoa through the perivitelline layer (PL) overlying the hen's ovum have been studied more frequently in the chicken than in other domesticated avian species. Species-specific action of the binding process was tested using an in vitro competition assay in which spermatozoa from the cock, tom, and drake were pretreated with solubilized PL protein (PL-P) from the chicken, turkey, and duck ovum. Spermatozoa were pretreated with PL-P for 20 min at 39 C and co-incubated in vitro with a .5 cm2 section of intact PL from the homologous sperm donor species for an additional 10 min at 39 C. Effectiveness of PL-P pretreatment was assessed quantitatively by the number of spermatozoa bound to the PL, and was expressed as a percentage of the control [minimum essential medium (MEM) pretreated sperm = 100%] binding. Pretreatment of cock spermatozoa with chicken, turkey, or duck PL-P resulted in 21, 40, and 48% binding, respectively. Similarly, pretreatment of tom spermatozoa with PL-P from chicken, turkey, or duck resulted in 45, 51, and 39% binding, and that of drake spermatozoa resulted in 38, 32, and 21% binding, respectively. Incubation of spermatozoa with PL-P from chicken, turkey, and duck ova indicated cross-reactivity and suppression of binding between avian spermatozoa and PL that was not species-specific.

Carbohydrate involvement in sperm-egg interaction in the chicken.

The solubilized perivitelline layer (PL) of the chicken ovum contains one or more components which behave in a manner analogous to sperm-receptors. Electrophoretic analyses of solubilized PL confirmed the existence of three major glycoproteins having apparent molecular weights of 33,000, 53,500 and greater than 200,000. The role of carbohydrate in sperm receptor activity was evaluated by extensive deglycosylation of the solubilized PL with trifluoromethanesulfonic acid (TFMS). Sperm receptor activity as measured by an in vitro competition assay was extremely sensitive to TFMS. Pretreatment of spermatozoa with solubilized PL exposed only to TFMS buffers inhibited sperm attachment and digestion of intact PL by 81% (9.67 +/- 1.76 sperm/mm2) as compared with the controls (i.e., no pretreatment of spermatozoa with solubilized PL; 51.33 +/- 6.24 sperm/mm2), a value similar to that observed with spermatozoa exposed to untreated solubilized PL (0.17 +/- 0.17 sperm/mm2). In comparison, PL treated with TFMS inhibited sperm attachment and digestion of intact PL by less than 26% (38.33 +/- 5.88 sperm/mm2) as compared with the control. These data indicate that removal of both N- and O-linked oligosaccharides from components of the chicken PL results in elimination of its sperm receptor activity.

Avian sperm-egg interaction: perivitelline layer possesses receptor activity for spermatozoa.

Sperm-egg interaction in mammals is initiated by the binding of spermatozoa to the zona pellucida (ZP), an acellular coat completely surrounding the vitelline membrane of unfertilized eggs. The perivitelline layer (PL) of the ovum of the hen is analogous to the mammalian ZP. In vitro incubation of the PL with spermatozoa results in fragmentation of the PL. The PL promotes the acrosome reaction whereby sperm acrosomal enzymes are released; the enzymes in turn dissolve the PL. The present study was conducted to determine if the PL removed from chicken ova, recovered immediately after ovulation, possess sperm-receptor activity. The results suggest that the solubilized PL does contain one or more components which behave in a manner analogous to sperm-receptors. Pretreatment of spermatozoa with solubilized PL prevented their subsequent attachment to and fragmentation of the intact PL which was observed with untreated spermatozoa.

Influence of neuraminidase on fertility and on sperm storage in the hen's oviduct.

Preinsemination incubation of semen with neuraminidase (4 IU/2 x 10(9) spermatozoa) significantly reduced fertility in one out of two trials. No differences were observed in fertility between semen treated with 4 versus 8 or 4 versus 16 IU of neuraminidase in Trials 1 or 2. All three levels of neuraminidase (4, 8, or 16 IU) removed the same amount (about 45%) of the bound sialic acids from spermatozoa during incubation. The removal of sialic acid from spermatozoa had a slight but nonsignificant affect on sperm storage within the uterovaginal (UV) sperm-host glands. Hens inseminated with neuraminidase-treated spermatozoa had decreased numbers of full and partially full UV sperm-host glands and increased numbers of empty glands, compared to hens inseminated with untreated but incubated spermatozoa. In the present study, reduced fertility resulting from the treatment of spermatozoa with neuraminidase from Clostridium perfringens indicates the desirability of exploring the use of other neuraminidases to see whether a correlation exists between the amount of sialic acid removed from spermatozoa and their subsequent fertility.

Fertility following intrauterine insemination near the time of oviposition.

The fate and fertility of semen deposited immediately following oviposition in the vicinity of the uterovaginal sperm host glands (USHG) was evaluated and compared with the semen deposited in the vagina. Intrauterine insemination of hens immediately after oviposition provided excellent fertility over a 2-wk period, compared to semen deposited in the vagina; also, 89% of the eggs laid within 25 h following intrauterine insemination were fertile, versus 33% following intravaginal insemination. Thus, spermatozoa deposited in the vicinity of the USHG immediately following oviposition (to simulate a release of spermatozoa from this region) can be transported to the infundibulum and effectively fertilize the next ovum ovulated. While some spermatozoa deposited in the uterus are transported to the infundibulum, the bulk of those retained in the oviduct populate the USHG. Thus, oviductal contractions immediately following oviposition do not adversely influence either the retention of spermatozoa or their incorporation into the USHG if the spermatozoa are placed on the uterine side of the uterovaginal junction.

Evaluation of buspirone as an antianxiety agent: buspirone and diazepam versus placebo.

Buspirone has previously been demonstrated to be efficacious in the treatment of anxiety. This four-week double-blind parallel study compared buspirone to diazepam and placebo in the treatment of 119 outpatients diagnosed as having generalized anxiety disorder. After a seven-day placebo washout period, eligible patients were randomized to one of three treatment groups. Buspirone (5 mg) and diazepam (5 mg) were administered BID and individually titrated to an optimal therapeutic dose by the end of week two. Buspirone and diazepam were equally effective in reducing Hamilton Anxiety (HAM-A) total and psychic factor scores from baseline values. Buspirone alone was significantly better than placebo in reducing the HAM-A somatic factor score. Sixty-seven percent of both active treatment groups who were classified as "ill" on the baseline global psychopathology rating scale achieved a "not ill" status by study end. There were no significant differences between treatment groups at endpoint on the 56-item Symptom Checklist self-rating scale. Buspirone was demonstrated to be as effective as diazepam in relieving anxiety in this outpatient sample.