Effects of Fluid Secreted from the Uterus on Duration of Fertile Egg Production in Hens, and Survivability and Penetrability of Fowl Sperm in vitro

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1 browse/ jpsa doi: / jpsa Copyright C 2013, Japan Poultry Science Association. Effects of Fluid Secreted from the Uterus on Duration of Fertile Egg Production in Hens, and Survivability and Penetrability of Fowl Sperm in vitro Muslah U. Ahammad 1, Tomoko Miyazato 2, Chiaki Nishino 2, Hideki Tatemoto 2, Nobuhiko Okura 3,ShinOkamoto 4, Yasuhiro Kawamoto 2 and Tadashi Nakada 5 1 Science of Bioresource Production, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima city, Kagoshima , Japan 2 Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa , Japan 3 Faculty of Medicine, University of the Ryukyus, Nishihara-cho, Okinawa , Japan 4 Faculty of Agriculture, Kagoshima University, Kagoshima city, Kagoshima , Japan 5 Professor Emeritus, Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa , Japan In this study, whether the secretion of the uterine fluid (UF) exerts a role in the prolongation of functional lifespan of the sperm in the hen oviduct was evaluated. The sperm were subjected to upper vaginal artificial insemination (AI) in the vicinity of the utero-vaginal junction (UVJ) under conditions of either non-secretion, plumping fluid (PF) secretion, or calcifying fluid (CF) secretion of the uterus of hens. Aliquots of the sperm were also subjected to in vitro storage at 41 for0,2,4,and6hinlake ssolution(ls),pfandcf. FollowingAI,theexposure of the sperm to either PF or CF secretion resulted in a remarkably a longer duration of the fertile egg production compared to when the AI was performed during the non-secretory phase of the uterus (P<0.05). Similarly, the in vitro exposure of the sperm to PF and CF during storage resulted in increased survivability and penetrability of the sperm compared with storage in LS(P<0.05). In particular, distinct functional differences (P<0.05) were observed between the sperm stored in vivo and in vitro under PF and CF conditions. The sperm inseminated in the presence of PF secretion exhibited a longer period of fertile egg production, and the sperm stored in PF in vitro exhibited a higher capacity for survival and penetration into the inner perivitelline layer (IPL) than those inseminated in the presence of CF secretion or were stored in vitro in CF, respectively, indicating that exposure of the sperm to the UF might influence sperm function. In conclusion, the in vivo and in vitro findings suggest that the secretion of UF in the oviduct of hens may prolong sperm survival and maintain the fertility potential of the fowl sperm. Key words: artificial insemination, fertility potential, sperm survival, uterine fluid J. Poult. Sci., 50: 74-82, 2013 Introduction The unique features of avian reproduction include the presence of specialized sperm storage tubules (SST) (Fujii and Tamura, 1963; Das et al., 2010) in the female oviduct and the prolonged storage of sperm in the SST without the loss of their viability and fertility. In fowl, studies have demonstrated that the sperm gradually acquire the capacity for survival and fertilization in the male reproductive tract during their passage from the testis to the distal vas deferens Received: April 3, 2012, Accepted: June 29, 2012 Released Online Advance Publication: August 25, 2012 Correspondence: Dr. Y. Kawamoto, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa , Japan. ( yasuk@agr.u-ryukyu.ac.jp) (Ahammad et al., 2011a, 2011b), whereas the oviduct provides an environment suitable for the resident sperm to retain their survival and fertility potential over an extended period (Yoshimura et al., 1997; Bré que et al., 2003; Bakst, 2011; Blesbois, 2012). In the oviduct, the SST that are typically located at the junction between the uterus and vagina, referred to as UVJ-SST, are the primary sites for sperm residence, and store the sperm more efficiently than the SST located in the infundibulum (Brillard, 1993; Bakst et al., 1994). It has been reported that during their long stay in the UVJ- SST, the sperm remain exposed to the PF and CF, commonly termed UF, secreted from the uterus during the plumping (continues for 5 h after the arrival of a membranous egg in the uterus) and calcifying (continues for 12 h after the ter-

2 Ahammad et al.: Uterine Fluid on Sperm Function 75 mination of the PF secretion) phases of secretion, respectively (Burke and Rieser, 1972). The secretion of the UF is known for its direct involvement in the formation of eggshell; however, studies of the biochemical composition of the PF and CF revealed that these fluids are composed of a complex mixture of inorganic salts and organic compounds, and they differ greatly in composition (Edward and Leach, 1980). The mineral and energy content of the PF and CF are within the range known to regulate motility and to maintain the metabolism of sperm, respectively. Importantly, these fluids also maintain an optimal ph, which stabilize the integrity of the sperm membrane. Thus, it is reasonable to hypothesize that the UF contributes to maintaining the survivability and fertilizing capacity of the sperm during storage in the UVJ-SST through providing a nutritive-protective aqueous milieu in the uterus. Until now, a large number of studies examined in detail the SST-related mechanisms behind the phenomenon of sperm longevity in the oviduct. In contrast, there are no studies investigating whether the exposure of sperm to the UF has biological significance for the prolongation of sperm storage in the UVJ-SST. Using AI to introduce sperm into the vagina of hens, Brillard et al. (1987) suggested that the fluid secreted from the uterus acts as a medium only for the transport of sperm in the oviduct, evidenced by the poor recovery rate of sperm in the oviduct at different intervals following AI in the absence of uterine secretory activity. However, the effects of the UF on maintaining the potential for the prolonged survival and fertilizing capacity of the sperm in the oviduct is unknown because the study did not investigate the effects of the PF and CF secretion on the sperm function in the oviduct. Therefore, to elucidate whether the UF plays a role in prolonging the functional life-span of fowl sperm in the hen oviduct, the ejaculated sperm were inseminated artificially into the upper vagina around the UVJ under plumping or calcifying secretion condition of the uterus to examine the effect of UF on duration of the fertile egg production. Because a number of intrinsic factors are involved in the process of prolonged storage of the sperm in the oviduct, this study also evaluated the direct influence of the UF on sperm function by investigating the ability of the sperm to survive and penetrate the IPL, a determinant of the fertility potential of the sperm, during the in vitro storage of the sperm in the PF and CF. Materials and Methods Animals Mature White Leghorn, Japanese Shamo (Fighting cock) and Ukokkei (Silky fowl) male chickens (Gallus gallus) aged 42 to 45 wks with proven fertility were used as semen donors in this study. Mature Single Comb White Leghorn female chickens aged wks were used in this study. Experimental birds were maintained individually in commercial laying cages under a photoperiod regimen of 14L:10D. Male birds were fed a commercial male breeder ration (19% crude protein (CP) and 2800 kcal metabolizable energy (ME)/kg) and female birds were fed a female breeder ration (16% CP, 2900 kcal ME/kg and 3.5% Ca) (Feed Production Unit, Uehara Poultry Farm, Itoman, Okinawa, Japan) ad libitum. This study was carried out in accordance with the regulations for the care and use of experimental animals prescribed by the Animal Care Committee of the University of the Ryukyus, Okinawa, Japan. Collection of the PF and CF Hens laying more than 5 eggs in a sequence, with a 1-d pause between clutches, for more than 3 wks prior to the experiments were selected as donors of the UF and for the AI. Approximately 5 h after ovulation, an ovum arrives in the uterus in a laying hen as a membranous egg, which acts as a stimulus for the secretion of the UF and stays in the uterus for a period of h (Warren and Scott, 1935). Immediately after the arrival of a membranous egg in the uterus, the PF continues to be secreted for 5-6 h to plump-up the membranous egg. After the termination of the PF secretion, the CF is continuously secreted from the uterus for h to build a hard shell around the outer membrane (Hoover and Smith, 1958; El Jack and Lake, 1967; Burke and Rieser, 1972; Nakada and Koga, 1990). The oviposition time (estimated by visualization) of the preceding egg and the time of arrival of the membranous egg in the uterus (estimated by palpation) were recorded for each individual hen at least two weeks prior to the collection of the PF and CF. Prior to the collection of the PF or CF from the uterus of a laying hen, the location of the membranous or shelled egg in the uterus was confirmed by palpation. A special stand, designed by Hoover and Smith (1958), was used to immobilize the hen. The collection of the PF and the CF was not performed simultaneously from the same laying hen. For 10 min, gravity was used to collect the PF in a glass tube, guided by a forefinger, using a multi-perforated bulb-shaped glass catheter that was placed inside the uterus. This procedure was performed 1 h post-arrival of a membranous egg in the uterus (Hoover and Smith, 1958). The same procedure was followed for the collection of the CF 13 h post-arrival of a membranous egg in the uterus. The PF samples and the CF samples were pooled and were centrifuged at 1500 g for 10 min to remove the cell debris and were stored 30 until use. Chemical Analysis of the PF and CF Immediately after centrifugation, the PF and CF supernatants were subjected to chemical analysis. The o-cresolphthalein complexion (OCPC) method was used to measure the calcium (Ca +2 ) content using a Wako Calcium C kit, Wako Pure Chemical Industries, Ltd., Osaka, Japan. The magnesium (Mg +2 ) and zinc (Zn +2 ) contents were estimated using the Mg-test Wako kit and the Zn-test Wako kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. The glucose content was measured using a Glucose CII-Test Wako kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). A glass electrode (D-52; Horiba, Kyoto, Japan) ph meter was used to measure the ph of the PF and CF. Semen Collection and Sperm Preparation Uncontaminated ejaculated semen devoid of transparent fluid was collected twice weekly by the dorso-abdominal

3 76 Journal of Poultry Science, 50 (1) massage technique described by Burrows and Quinn (1935) from those selected adult roosters and pooled. Immediately after collection, the pooled semen was diluted at a rate of 1 part semen to 3 parts LS, ph 7.1 (Lake, 1960), PF or CF and washed two times by means of centrifugation at 600 g for 10 min to discard seminal plasma. The sperm pellets were then resuspended in the corresponding diluents. Sperm suspended in LSwere subjected to AI into the hens and storage at 41 for 0, 2, 4, and 6 h under in vitro conditions, whereas sperm suspended in PF and CF were subjected to storage under the same conditions. The number of sperm cells was counted using a Neubauer hemocytometer (American Optical Co., New York, NY, USA). The final concentration of sperm was adjusted to cells/ml and the diluted sperm suspensions thus obtained were divided into series of aliquots for use in AI. AI of Hens and Determination of Duration of Fertile Egg Production For the AI, a total of 39 experimental hens were selected randomly from those hens that were used as donors of the UF. Hens were divided into three groups (n=13/uterine phase) according to the phases of the uterus and were subjected to a single AI directly into the upper vagina around the UVJ with sperm (Brillard et al., 1987) in a 0.2 ml dose of diluted semen per hen during either non-secretory (1 h after oviposition), PF secretory (1 h after the arrival of a membranous egg in the uterus) or CF secretory (13 h after the arrival of a membranous egg in the uterus) phase of the uterus. The expected time of the arrival of a membranous egg in the uterus was calculated from the record of oviposition of the preceding egg based on the standard time required for the process of an egg formation in the hen oviduct (Warren and Scott, 1935). In addition, prior to AI, the presence of an egg in the uterus was ascertained by palpation. A rubber inseminating catheter with blunt tip fitted with a disposable graduated syringe was placed inside the upper vagina, guided by the forefinger of the left hand covered with latex gloves, for upper vaginal AI of the sperm. Eggs were collected two times daily from the second day after AI, and then incubated (37.8 temperature, 60-65% relative humidity, <0.5% CO 2 ) in a forced-draft automatic incubator (Toyo Incubator TS15, Ashida Sangyo Co., Ltd., Okayama, Japan). Fertility ((number of fertilized eggs/total eggs set) 100) was determined by candling on day 7 of incubation. Eggs thought to be infertile were broken open and examined the germinal disc region macroscopically for evidence of embryonic development. Following a day after the AI, the duration of production of the fertile eggs was determined, according to the definition given by Brillard et al. (1989), as the number of days from the day after a single AI to the day before oviposition of the last fertile egg. Observation of Sperm in the SST At 24 h post-ai, 3 hens were selected randomly from each uterine phase group and were killed by the rapid intravenous injection of a lethal dose of sodium pentobarbital (Nembutal; Dainippon Sumitomo Pharmaceutical, Osaka, Japan). This 24-h timeframe was considered sufficient to ensure the maximal filling of the SST of the hens (Brillard, 1993). The preparation of the UVJ and the infundibular mucosa for the observation of the SST was performed following the procedure described by Ito et al. (2011). Briefly, following laparotomy, the oviduct was removed from the body cavity and the UVJ and infundibulum were excised longitudinally using a fine scissors and a forceps. The mucosal layer of each UVJ and infundibulum was scraped using a scalpel. The individual mucosal folds containing the SST either from the UVJ or the infundibulum were isolated and incubated in phosphate buffered saline (PBS; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 0.1% Triton X-100 at room temperature for 10 min, followed by washing with PBS. The specimens were fixed overnight at 20 in a 1:1 (v/v) mixture of acetone and methanol. After a final wash with PBS, the fixed specimens were subjected to fluorescence staining using 4, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) for 10 min and were examined under a fluorescence microscope (Nikon Eclipse 50i, Nikon, Tokyo, Japan). At least 100 SST were examined, and the number of SST containing sperm (Fig. 1) was counted to calculate their filling rate (%). Assessment of Sperm Survival in vitro To determine whether UF maintains survivability of the sperm at the body temperature of hens (41 ) under in vitro conditions, aliquots of the sperm were stored either in PF or CF at 41 for 0, 2, 4, and 6 h. Storage of the sperm in LS under the same conditions served as a control. At the end of each storage period, the sperm were examined for their live/dead status. Viability of the sperm was assessed using live-dead dual fluorescent staining with SYBR-14 (1 mg/ml anhydrous dimethyl sulphoxide (DMSO) (Sigma-Aldrich, Corp., St. Louis, Missouri, USA) and propidium iodide (PI) (2 mg/ml PBS) (Invitrogen, Inc., Eugene, OR, USA), as previously described by Chalah and Brillard (1998). Briefly, 500 μl sperm suspension at a concentration of cells/ Fig. 1. Digital micrograph showing the sperm storage tubules (SST) containing resident sperm detected by fluorescence staining with 4, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) under a fluorescence microscope. Scale bar=20 μm at 200 magnification.

4 Ahammad et al.: Uterine Fluid on Sperm Function 77 Fig. 2. Digital micrograph showing the formation of penetration holes on the surface of the inner perivitelline layer (IPL) by sperm during a 30-min period of incubation with IPL at 39 under in vitro condition. Scale bar=10 μm at100 magnification. ml of each aliquot was mixed with 4 μl of SYBR-14 (green fluorescent)/pi (red fluorescent) and incubated for 10 min at room temperature. At least 150 sperm per replication were observed at 400 magnification under a fluorescence microscope equipped with blue illumination (excitation at nm, emission at nm). Sperm were categorized as live or dead when they exhibited complete green fluorescence or red fluorescence, respectively. Collection of the IPL The IPL was separated from the outer perivitelline layer (OPL) by acid hydrolysis using a day-old oviposited chicken egg as described by Kido and Doi (1988) for use in sperm penetration test. Briefly, the egg was broken open, and the albumen was drained. The yolk was placed in a beaker containing 60 ml of 0.01 M HCl and incubated at 37 for 1 h. After incubation, the yolk was punctured using blunt forceps, and the perivitelline layer (IPL+OPL) was pulled up and washed for 30 s in the same HCl. The roughly washed perivitelline layer was again washed several times in 1% NaCl (w/v) to remove the yolk material completely and then spread out in a glass Petri dish containing 1% NaCl (w/v). The IPL was pulled from the OPL under a stereomicroscope. Sperm Penetration Test To examine the capacity of the sperm for penetration into the IPL following 0, 2, 4, and 6 h storage of the sperm either in LS, PF or CF, a sheet (0.5 cm 0.5 cm) of intact IPL was incubated with a total of sperm cells at 39 for 30 min in an incubation tube (Sorensen, Bioscience, Inc., Japan) containing 1 ml MEM according to the method followed by Robertson et al. (1998). After incubation, the IPL was immersed in 3% NaCl (w/v) to stop the interaction between the IPL and sperm, mounted on a microscope slide and stained with Schiff s reagent (Sigma-Aldrich, Corp., St. Louis, Missouri, USA) for 10 min following fixation with 20% (v/v) formalin for 20 s. Successful sperm penetration was indicated by the formation of a hydrolyzed hole (Fig. 2) in the IPL (Bakst and Howarth, 1977; Okamura and Nishiyama, 1978). The number of all visible holes per 0.25 mm 2 was counted at 100 magnification with a digital micro counter (Digital Sight DS-L2, Nikon, Tokyo, Japan) attached to a light microscope. Statistical Analysis Data from six replicate trials were expressed as the mean ±SEM. Percentage data were subjected to arcsine transformation to satisfy the normality assumption for the analysis of variance (ANOVA) prior to statistical analysis. All statistical analyses were performed using the Statistical Analysis System R software package (R Development Core Team, 2008). Data were analyzed by ANOVA using the generalized linear model (GLM) procedure. In cases where analyses revealed a significant treatment effect, differences between mean values were evaluated using the Bonferroni multiple comparison test. A value of P<0.05 was considered statistically significant, unless stated otherwise. Results Chemical Composition of the PF and CF The PF and CF were not collected simultaneously from the same hen. Distinct variations (P<0.05; Table 1) were observed in the chemical compositions of the PF and CF, which were collected from the uterus at 1 h and 13 h after the arrival of a membranous egg in the uterus, respectively. The mean volume of the PF secreted from the uterus was approximately two times greater than the volume of CF secreted. The ph was also higher in the PF compared with the CF. In contrast, the concentrations of Ca +2,Mg +2,Zn +2 and glucose were almost three, six, one and a half and two times higher, respectively in the CF than in the PF (Table 1). Filling Rate of the SST When a given number of sperm was inseminated into the upper vagina around the UVJ-SST of hens during the nonsecretory, PF secretory or CF secretory phase of the uterus, the filling rates of the UVJ-SST were minimum, medium and maximum for the AI performed during the non-secretory, CF secretory and PF secretory phases of the uterus, respectively (P<0.05; Fig. 3). As shown in Fig. 3, irrespective of the different phases of the uterus, the number of SST containing sperm was higher (P<0. 05) in the UVJ-SST than in the infundibular SST. The mean (±SEM) filling rate (%) of the UVJ-SST for the AI performed during the non-secretory, PF secretory and CF secretory phases of the uterus were 31.6±2.8, 66.5±1.6 and 52.0±2.2, respectively (Fig. 3). In contrast, the mean (± SEM) filling rate (%) of the infundibular SST for the AI performed during the non-secretory, plumping secretory and calcifying secretory phases of the uterus were 18.4±2.1, 33.1±2.5 and 39.2±1.9, respectively (Fig. 3). Duration of the Fertile Egg Production Considering the effect of different phases of the uterus on the duration of the fertile egg production from the second day post-ai of the sperm into the upper vagina of the hen, different (P<0.05; Fig. 4) periods of fertilized egg production

5 78 Journal of Poultry Science, 50 (1) Table 1. Volume and chemical composition of the plumping and calcifying fluid collected from the uterus at 1 h and 13 h after the arrival of a membranous egg in the uterus of laying hens Parameters Volume (ml/hen) Calcium (Ca +2 ; mmol/l) Magnesium (mg +2 ; mmol/l) Zinc (Zn +2 ; μg/dl) Glucose (g/l) ph Number of samples Plumping fluid 8.4±1.7 a 3.3±0.1 a 2.4±0.6 a 3.6±0.2 a 4.2±0.1 a 7.7±0.4 a Calcifying fluid 4.3±1.5 b 9.4±0.3 b 13.6±0.8 b 4.1±0.3 b 7.9±0.1 b 7.5±0.1 b Significant difference between the two groups (plumping fluid and calcifying fluid) was evaluated by the Student s t-test. a,b Values without a common superscript within the same row differed (P< 0.05) between plumping and calcifying fluids of the uterus. Fig. 3. Filling rate (%) of the sperm storage tubules (SST) after artificial insemination of sperm into the upper vagina around the utero-vaginal junction (UVJ) under conditions of non-secretion, plumping fluid (PF) secretion and calcifying fluid (CF) secretion of the uterus of laying hens. Data are expressed as the mean±sem (n= 6). * Values differed (P<0.05) between the UVJ-SST and infundibular SST sources as judged by two-way ANOVA with GLM. Differences between mean values within the phases of the uterus were evaluated using Bonferroni multiple comparison test after analysis of data by oneway ANOVA with GLM. a-c Values without a common superscript differed (P<0.05) among uterine phases of non-secretion, PF secretion and CF secretion. were observed for the sperm inseminated during the nonsecretory, PF secretory and CF secretory phases of the uterus. As shown in Fig. 4, the sperm placed directly into the upper vagina under the conditions of non-secretion, PF secretion and CF secretion of the uterus resulted in the minimal (13.3 ±1.9 d), maximal (24.2±1.4 d) and medium (19.5±1.8 d) durations of production of the fertile eggs, respectively. Survivability of Sperm Stored in vitro The mean (±SEM) percentage of viable sperm, detected by the live-dead dual fluorescent staining with SYBR-14/PI, in the PF (86.6±2.2) or in the CF (86.4±1.3) did not differ (P>0. 05, Fig. 5) from storage in LS(87. 1±1. 5) at the initiation of the storage in vitro at 41 (the body temperature of hens). However, the viability of the sperm, irrespective of the diluents used for storage, decreased with the prolongation of the storage time. As shown in Fig. 5, the sperm stored in vitro in LSat 41 survived for up to 2 h during which the proportion of viable sperm was 56.4±2.3 %, whereas storage of the sperm in the PF for 4 h and in the CF for 2 h under the same conditions resulted in no apparent

6 Ahammad et al.: Uterine Fluid on Sperm Function 79 Fig. 4. Duration of the fertile egg production after upper vaginal artificial insemination of the sperm during either non-secretory, plumping fluid (PF) secretory or calcifying fluid (CF) secretory phase of the uterus of laying hens. Data are expressed as the mean±sem (n= 6). Differences between mean values of duration of the fertile egg production within the phases of the uterus were evaluated using Bonferroni multiple comparison test after analysis of data by one-way ANOVA with GLM. a-c Values without a common superscript within the same column differed (P<0.05) among non-secretory, PF secretory and CF secretory phases of the uterus. loss (P>0. 05) of their initial viability. Viability of the sperm stored in the PF and CF was then declined (P<0.05) thereafter. However, at 4 and 6 h of storage periods, the sperm stored in the PF exhibited a higher (P<0.05; Fig. 5) survival rate than the sperm stored in the CF. As shown in Fig. 5, the mean percentage of the sperm that survived for a 4-h period of storage was 81.7±1.8 and 70.5±2.1 in the PF and CF, respectively, and during the 6-h period of storage, the mean percentage survival was 69.4±2.7 and 62.2±1.9 in the PF and CF, respectively. Therefore, after 4 h of storage, the decrease in the proportion of viable sperm stored in the PF and CF, calculated from their corresponding initial values, was 5.7 and 18.4%, respectively, and 19.9 and 28.0%, respectively, after 6 h of storage. Penetrability of Sperm Stored in vitro The capacity of the sperm to produce holes in the IPL was examined in vitro after storage of the sperm in LS, PF and CF at 41 for 0, 2, 4, and 6 h. As shown in Fig. 6, immediately after storage (0 h), no differences (P>0.05) in the ability of the sperm stored in LS, PF and CF to penetrate the IPL were observed (assessed by the number of penetration holes formed per 0.25 mm 2 area in the IPL during a 30-min interaction of the sperm with the intact IPL). However, the number of holes produced in the IPL by the sperm, irrespective of the diluents used for storage, decreased as the length of the storage time increased. The sperm stored for 2 h in LSproduced the least number of holes (30.5±3.6); they lost 51.0% of their initial penetrability. In contrast, no significant re- Fig. 5. The mean proportion of viable sperm, detected by SYBR-14/propidium iodide (PI) dual fluorescent staining, after storage of the sperm in Lake s solution (LS), plumping fluid (PF) and calcifying fluid (CF) at 41 for 0, 2, 4, and 6 h under in vitro conditions. Data are expressed as the mean±sem (n=6). Differences between mean values within the diluents of sperm were evaluated using Bonferroni multiple comparison test after analysis of data by one-way ANOVA with GLM. a-c Values without a common superscript differed (P<0.05) among sperm samples stored in LS, PF and CF at each of the storage periods. NS Values did not differ (P>0.05; Student's t-test) as compared with the control (0 h of storage) in the same diluent. * Values differed (P<0.05; Student s t-test) as compared with the control (0 h of storage) in the same diluent.

7 80 Journal of Poultry Science, 50 (1) Fig. 6. The mean numbers of holes on the surface of the inner perivitelline layer (IPL) as an indication of penetration into the IPL by sperm after storage in Lake s solution (LS), plumping fluid (PF) and calcifying fluid (CF) at 41 for 0, 2, 4, and 6 h under in vitro conditions. Data are expressed as the mean± SEM (n=6). Differences between mean values within the diluents of sperm were evaluated using Bonferroni multiple comparison test after analysis of data by oneway ANOVA with GLM. a-c Values without a common superscript differed (P< 0. 05) among sperm samples stored in LS, PF and CF at each of the storage period. NS Values did not differ (P>0.05; Student s t-test) as compared with the control (0 h of storage) in the same diluent. * Values differed (P<0.05; Student s t-test) as compared with the control (0 h of storage) in the same diluent. duction in the formation of penetration holes was observed for the sperm stored in the PF and CF for 2 h. However, after storage in the PF and CF for 4 h, the sperm produced 51.0± 2.9 and 44.3±3.1 holes, respectively; therefore, the sperm retained 82.9 and 71.3% of their initial penetrability, respectively. At 4 and 6 h of storage periods, the sperm stored in the PF exhibited higher (P>0.05; Fig. 6) ability to produce penetration holes in the IPL than the sperm stored in the CF. The mean numbers of holes produced by sperm stored in the PF and CF were declined from their initial corresponding values of 61.5±3.4 and 62.1±2.6 to 38.5±2.9 and 31.3± 2.7, respectively, during the 6-h period of storage. Discussion In this study, compared to performing the AI during the non-secretory phase of the uterus, the number of SST that were filled with sperm increased significantly when the AI was performed during either PF or CF secretory phase of the uterus. The filling rate of the UVJ-SST was significantly higher following the AI of sperm during the plumping phase of UF secretion than the AI of sperm performed during the calcifying phase of UF secretion. It is known that the higher the number of sperm stored in the SST, the longer the duration of production of the fertile eggs (Zavaleta and Ogasawara, 1987; Brillard, 1993). As expected, coinciding with the significantly different filling rates of the SST observed in this study, the durations of the fertile egg production were minimum, medium and maximum for the AI of sperm into the upper vagina during the non-secretory, the CF secretory and the PF secretory phases of the uterus, respectively. These data imply that the secretory conditions of the uterus at the time of the upper vaginal AI have a significant effect on the duration of the fertile egg production. This finding is in agreement with other studies reporting a longer period of fertilized egg production by hens when the AI is performed in the afternoon or evening compared to in the morning (Johnston and Parker, 1970; Brillard et al., 1987). The duration of the fertile egg production following the AI of fowl sperm into the hen oviduct is used widely to assess the length of sperm survival in the oviduct. However, in this study, because the number of SST containing sperm differed significantly in the three experimental groups, the survivability of the sperm stored in the SST after upper vaginal AI during the non-secretory, PF secretory and CF secretory phases of the uterus cannot be explained by the different periods of the fertile egg production. Therefore, the influence of the UF on sperm survival was evaluated in vitro by storing the sperm directly in PF and CF at the hen s body temperature of 41. This study provided direct evidence that when the sperm were stored in LS, more than half of the sperm lost their viability within 2 h at 41. In contrast, the viability of the sperm was not affected significantly by storage in PF or in CF for 4 h, and most of the stored sperm remained viable during a 6-h storage period, indicating that the UF provides

8 Ahammad et al.: Uterine Fluid on Sperm Function 81 an environment suitable for the maintenance of sperm survival. Although the phenomenon of prolonged survival of sperm in the oviduct is completely different from the phenomenon of short-term storage of sperm in vitro, the UF exhibits a significantly higher capacity to store sperm for 4 h without affecting survivability compared to the storage ability of LS. This observation implies that the UF provides a nutritive-protective environment for the sperm, which is conducive to better survival. The results of this study also provided in vitro evidence that the exposure of sperm to PF resulted in significantly higher survivability compared to storage in CF. The reasons for the differential influence of PF and CF secretions on sperm survival are complex. The chemical constituents of the UF secretions appear to be effective for maintaining the survival potential of the sperm by stabilizing the sperm membranes. The presence of magnesium (Mg +2 ), zinc (Zn +2 )and glucose in the diluents reduces the oxidative damage of the sperm plasma membrane, depresses the sperm metabolism and supplies energy to the sperm for metabolism, respectively (Fujihara and Koga, 1984; Bakst, 1985). Because the CF contained significantly higher concentrations of magnesium, zinc and glucose than the PF, as reported in this study, the lower survivability of the sperm in the CF seems unlikely. However, the exceptionally high calcium content in the CF may stimulate sperm motility in the CF, thereby accelerating the oxygen consumption by the sperm that causes hypermetabolism in the sperm leading to the production of toxic metabolic by-products in the storage medium. In this study, we examined further the capacity for penetration into the IPL by the sperm after storage in PF and CF for various periods to evaluate whether UF can sustain fertility potential of sperm in vitro. Thein vitro study demonstrated that the capacity of the sperm to penetrate the IPL was affected adversely by storing the sperm in LS, whereas the sperm stored in either PF or CF retained the capacity of the sperm to penetrate the IPL, implying that the UF maintains the sperm potential for fertilization. It is known that extracellular Ca +2 in the storage medium induces 5-10% of the stored sperm to undergo an acrosome reaction within 5 min at 41 (Lamoine et al., 2008). Despite the high Ca +2 content, the mechanism by which the UF maintained the high fertility potential of the sperm for the prolonged storage period in the oviduct and during a 6-h in vitro storage in liquid, is unclear. However, it has been reported that the alkaline ph of the diluents is significant for stabilizing the acrosomal membrane integrity, thus sustaining the fertility potential of the sperm (Lake and Ravie, 1979). In conclusion, the findings of the in vivo and in vitro studies suggest that the UF plays a pivotal role in the prolongation of duration of the fertile egg production, sperm survival, and in the maintenance of the capacity of the sperm to penetrate the IPL. Acknowledgments We would like to thank Mr. Miyagi Tateo for providing us with Silky fowl from his breeding stocks. We are also grateful to Mr. Uehara Hajime for supplying White Leghorn cockerels and hens on a regular basis from his Poultry Farm (Breeding Unit). References Ahammad MU, Nishino C, Tatemoto H, Okura N, Okamoto S, Kawamoto Y and Nakada T. Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract. Theriogenology, 76: a. Ahammad MU, Nishino C, Tatemoto H, Okura N, Okamoto S, Kawamoto Y and Nakada T. 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