Effects of Frequency of Semen Collection on Quantitative and Qualitative Characteristics of Semen in Turkey Breeder Males 1

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1 Effects of Frequency of Semen Collection on Quantitative and Qualitative Characteristics of Semen in Turkey Breeder Males 1 J. NOIRAULT and J. P. BRILLARD2 Institut National de la Recherche Agronomique, Station de Recherches Avicoles, Centre de Tours, Nouzilly, France ABSTRACT The effects of various frequencies of semen collection on several quantitative and qualitative semen characteristics were investigated in adult turkey breeder males (30 to 40 wk of age). In Experiment 1, a total of 35 males were first trained for semen collection (twice a week for 2 consecutive wk), and then divided into five groups (seven males each), each group being collected either once every 2 wk, once every week, twice every week, three times every week (each for 4 wk) or five to seven times per week (each for 2 wk). Volume, sperm concentration, and sperm number per ejaculate were determined for each ejaculate. No significant differences between groups were observed for sperm concentration (P > 0.05), but males collected once every 2 wk, once per week, or twice per week had larger volumes than males collected at higher frequencies (P < 0.05). Thus there were significant differences for sperm number per ejaculate between groups (P < 0.05). Also, daily semen output (DSO) was markedly increased in males collected at the highest frequencies (e.g., DSO = and in males collected once and five times per week). Finally, in euthanatized birds (36 wk) no differences between groups were observed for body weight (25.8 ± 1.7 kg), testicular weight (51.5 ± 2.2 g), or total number of elongated spermatids per male (14.0 ± ). In Experiment 2, 35 males were distributed into groups and collected under the same conditions as in Experiment 1. Besides quantitative analyses of ejaculates (volume, sperm concentration, and sperm per ejaculate), sperm viability between groups was also tested using the Sybr14/PI fluorescence test. Our results demonstrated: 1) a favorable effect of high semen collection frequencies on sperm viability and, 2) a marked decline in sperm viability during the first 2 d following a 2-d resting period in males collected five times a week. We concluded that turkey males express their optimal reproductive capacity more efficiently when semen collection is undertaken at a high rather than a low frequency. (Key words: turkey, semen, sperm output, viability, artificial insemination) 1999 Poultry Science 78: INTRODUCTION The absence of adequate procedures to handle poultry semen over long periods and the interest of using the best genetic potential available from male line sires are two major reasons to optimize semen collection frequency in artificially inseminated flocks. Generally, semen is collected from turkey males once or twice a week in most commercial operations, despite the absence of physiological bases to support this pattern. Previous studies on the effects of semen collection frequency with respect to quantitative traits generally indicate an inverse relationship between ejaculate volume and frequency of semen collection, whereas sperm concentrations remain unaffected (Lorenz et al., Received for publication November 9, Accepted for publication February 25, This research was supported by a cooperative research partnership with CIDEF (Comite Interprofessionnel de la Dinde en France). 2To whom correspondence should be addressed: brillard@tours. inra.fr 1955; McCartney et al., 1958; Cecil et al., 1988). Moreover, some indications exist in the above publications that changes in the frequency of semen collection do not affect fertility traits in artificially inseminated hens (McCartney et al., 1958; Cecil, 1982). These results indirectly contradict in vitro observations by Brown (1968) that increasing the semen collection frequency significantly reduces the percentage of abnormal spermatozoa in turkey ejaculates. From a theoretical standpoint, the number of semen doses available per week per male rather than the number of doses per ejaculate should be considered to quantitate the final efficacy of semen collection protocols as this provides an optimal response to determine male to female ratios in breeder flocks. The present study was carried out to evaluate the consequences of various semen collection frequencies (from once every 2 wk to once per day) on several quantitative characteristics of turkey semen (volume, concentration, sperm per ejacu- Abbreviation Key: DSO = daily sperm output. 1034

2 SEMEN OUTPUT IN MALE TURKEYS 1035 late) and also to determine their possible effects on the viability of freshly ejaculated spermatozoa. MATERIALS AND METHODS Animal Husbandry and Semen Collection Experiment 1. Thirty-five young adult breeder males (BIG6 medium, British United Turkeys, 30 wk old), raised as recommended in the breeder s guide, were used for this study. Briefly, they were placed in floor pens from 21 wk of age and supplied with ad libitum access to water and a standard 10% protein diet until the end of the experiment (36 wk). From 23 wk of age, all birds were submitted to a 14-h photoperiod (provided by incandescent bulbs) with light intensity adjusted to 25 lx at the height of their heads. They were divided into five groups of seven males each at 30 wk. Twice weekly semen collection was then initiated up to 32 wk of age (training period) using the massage technique previously described by Burrows and Quinn (1937). After the training period, during which no differences were observed between groups for semen volume and semen concentration, each group then underwent the following schedule of semen collection for 4 consecutive wk: Group 1, once per 2 wk (Wednesday); G 2, once per week (Wednesday); G 3, twice per week (Monday and Friday); G 4, three times per week (Monday, Wednesday and Friday); or G 5, daily for 12 consecutive d, then a 2-d rest period, then five times per week for 2 consecutive wk. All males were weighed after the last semen collection and then killed (overdose of pentobarbital), the testes were removed and weighed (nearest milligram) and one fragment (approximately 1 g each, also weighed to nearest milligram) of each testis was ground in sucrose (0.25 M) as described by de Reviers (1972). The populations of elongated spermatids plus testicular spermatozoa were estimated using Thoma hemacytometers (six replicates per fragment). Experiment 2. This experiment was conducted with similar numbers of males and environmental conditions to those in Experiment 1, except that the training (2 wk) and experimental (4 wk) periods were undertaken between 33 and 39 wk of age. All ejaculates collected during the experimental period were examined for their quantitative characteristics (volume, concentration, sperm per ejaculate) and sperm viability. Quantitative Characteristics of Ejaculates Ejaculates were collected individually in 5-mL polypropylene tubes (two strokes per male: Bakst and Cecil, 3Molecular Probes Inc., Eugene, OR OSI, Paris, France Abacus Concept Inc., Berkeley, CA a,b). Ejaculate volumes were estimated with a scale to the nearest milligram, assuming that specific gravity of turkey semen is g/cm3. Previous observations in our laboratory indicated that at extremes, specific gravity of turkey semen varies between and It therefore appeared unnecessary to perform individual corrections between males or ejaculates, because using specific gravity on the basis of g/cm3 led to a maximum relative error 1%. For each ejaculate, sperm concentration was indirectly estimated with a Jenway colorimeter (wavelength: 540 nm, dilution of semen aliquots 1:200 vol/vol in saline; two aliquots per sample) by converting optical density in sperm concentration using a preestablished conversion table (unpublished data). These measurements made it possible to estimate total numbers of sperm per ejaculate. Comparisons between groups per time unit were performed on a per day basis (Daily Sperm Output or DSO), as proposed by Amann (1970). Briefly, DSO can be obtained from the formula: total number of spermatozoa collected during 1 wk/7 (Brillard and de Reviers, 1985). Sperm Viability (Experiment 2) Immediately after semen collection, one aliquot of each ejaculate was added to Lake s diluent (Lake and Ravie, 1982) up to a final volume of 500 ml and a final concentration of sperm per milliliter. Sperm viability analysis was performed for each diluted sample using a live-dead sperm viability kit (Donoghue et al., 1995; Chalah and Brillard, 1998). This commercially available test3 is an association between two nuclear fluorescent probes, one being propidium iodide, which stains the nucleus of nonviable spermatozoa in red, and Sybr14, a counter stain that reveals the nucleus of viable cells in bright blue. The preparations were examined with a BHS Olympus fluorescence microscope4 adjusted to 500 magnification. A total of two separate countings of 300 sperm (randomly chosen from two slides: 300 sperm per slide) were classified as viable or nonviable for each ejaculate. Statistical Analyses Comparisons between groups were performed using a single classification ANOVA and Fisher protected least significant difference (PLSD) after an ad hoc test when appropriate (P < 0.05, ANOVA). Statview 4.01 software5 for Apple MacIntosh computers was used for these analyses. RESULTS Quantitative Analysis of Ejaculates (Experiment 1) Comparison of the mean values of ejaculate volume, sperm concentration, sperm per ejaculate, and DSO

3 1036 NOIRAULT AND BRILLARD TABLE 1. Quantitative characteristics of ejaculates collected from turkeys (n = 7 per group) submitted to various frequencies of semen collection (x ± SE) Frequency Concentration Volume Tot. sperm/ejac DSO 1 ( 10 9 sperm/ml) (ml) ( 10 9 sperm) ( 10 9 sperm) Once every 2 wk ± 0.15 a 0.44 ± 0.12 a 4.29 ± 0.72 a 0.32 ± 0.05 e Once per week ± 0.35 a 0.43 ± 0.12 a 4.32 ± 0.74 a 0.62 ± 0.11 d Twice per week ± 0.24 a 0.42 ± 0.10 a 4.00 ± 0.37 a 1.14 ± 0.11 c Three times per week ± 0.27 a 0.37 ± 0.09 b 3.55 ± 0.26 b 1.53 ± 0.12 b Five times per week ± 0.36 a 0.27 ± 0.08 c 2.70 ± 0.17 c 1.93 ± 0.13 a Seven times per week ± 0.51 a 0.25 ± 0.02 c 1.82 ± 0.09 d 2.03 ± 0.19 a a emeans within a column with no common superscript differ significantly (P < 0.05). 1Daily sperm output. 2Four consecutive weeks. 3Two consecutive weeks. between groups of males (Experiment 1) is reported in Table 1. When compared at ejaculate levels, males subjected to collection most frequently (i.e., at least three times a week) had smaller semen volumes (0.25 to 0.37 ml) than males that had semen collected less often (0.42 to 0.44 ml; P < 0.05). By contrast, no significant differences were observed between groups of males for sperm concentrations, with values ranging from 9.82 to sperm per milliliter. The lowest mean number of sperm per ejaculate was observed in males that had semen collected once per day ( sperm) and the highest in males that had semen collected once every 2 wk and once a week (4.29 and sperm, respectively). When expressed in terms of DSO, the mean numbers of spermatozoa collected per male in each group gradually increased with the frequency of semen collection, with maxima reaching from 0.32 to sperm in males that had semen collected from once every 2 wk (Group 1) to once every day, respectively (Group 5; also see Figure 1). Finally, a collect-to-collect comparison of sperm per ejaculate in Group 5 (Figure 2) clearly indicated a marked day effect of semen collection during the days immediately following a resting period of either 3 d just before Period 1 or 2 days just before or during Period 2. More precisely, DSO significantly declined (P < 0.05) over time during the first 2 to 3 d following each resting period before maintaining baseline levels up to the end of the period. Testicular Sperm Production (Experiment 1) A series of comparisons of testicular weights, number of spermatozoa per gram of testis, and number of spermatozoa per male between groups of males is reported in Table 2. None of these criteria varied significantly between groups (P > 0.05). Qualitative Analyses of Spermatozoa (Experiment 2) The levels of DSO obtained from males submitted to the same frequency of semen collection were comparable between Experiments 1 and 2, irrespective of the frequency (P > 0.05). In addition to the mean values of DSO per group in Experiment 2, the percentages of viable spermatozoa according to the frequency of semen collection are presented in Table 3. These results indicated a significant increase in the mean percentage of viable spermatozoa in males collected more frequently, with values reaching 85.2% and 89.7% in Group 1 and Group 5, respectively (P < 0.05). Overall, the above results allowed the calculation of a mean viable DSO in each group, which indicated that the mean number of viable spermatozoa per group collected from males in Groups 1 (once every 2 wk) to 5 (five times per week) varied in a ratio of 1 to 6.9. When expressed on a per day basis during the days following a resting period (see Materials and Methods), a general tendency for increased viability of spermatozoa over time was observed, with significant differences (P < 0.05) between days within a week in Group 5 (Table 4). FIGURE 1. Mean values of daily sperm output (DSO) in turkey breeder males collected at various frequencies. Means with no common letter differ significantly (P < 0.05). DISCUSSION The relative advantages and disadvantages following high and low frequencies of semen collection have been

4 SEMEN OUTPUT IN MALE TURKEYS 1037 FIGURE 2. Day-to-day evolution of sperm output in turkey breeder males collected either seven times per week or five times per week. Means with no common letter differ significantly (P < 0.05). well documented in fowl by McDaniel and Sexton (1977). In the absence of a definite effect on fertility, and given that fowl hens are inseminated at weekly intervals, these authors concluded that, by influencing the number of spermatozoa available per day per week, the frequency of semen collection in that species also influences the number of semen doses available for insemination. Our observations in turkeys partly confirm the finding of previous studies that increasing the frequency of semen collection first increases semen yields per male. In the present study, DSO varied in a ratio of 1 to 3.4 between males collected one to five times weekly. In agreement with previous studies in turkeys (McCartney et al., 1958; Cecil, 1982), increasing the frequency of semen collection had negative effects TABLE 2. A comparison of testis weights and testicular populations of spermatozoa between males (n = 7 per group) initially submitted to various frequencies of semen collection (x ± SE) Total Testis weight Sperm per gram of testis number of testicular sperm per Frequency (total/male) ( 10 6 ) male ( 10 9 ) Once every 2 wk ± ± ± 2.41 Once per week ± ± ± 1.64 Twice per week ± ± ± 1.22 Three times per week ± ± ± 1.51 Five times per week ± ± ± Four consecutive weeks. 2Two consecutive weeks. (g) on ejaculate volumes without significantly affecting sperm concentrations, at least when semen collections were performed on a daily basis. However in our work, an explanation for the absence of significant differences in sperm concentrations between ejaculates from males subjected to collection at varying frequencies may be the relatively small number (seven) of males per treatment due to the high interindividual variability of semen parameters between males within groups. Moreover in the present study, both testicular weights and populations of elongated spermatids plus testicular spermatozoa per gram of testis were comparable between groups of males at the end of the semen collection period (P > 0.05 in both cases). These results partly contradict the earlier findings of Cecil et al. (1988), in which moderate (± 24%) but significant differences in spermatozoal reserves (P < 0.05; estimated at the testicular level) were observed between groups of males either subjected to collection daily or rested for a 3-wk period. Our results demonstrate that, following a rest period of 2 d or more, 3 d of daily semen collection are necessary for turkey males to reach their DSO base level (approximately 1.6 to spermatozoa per ejaculate). These results are well above those observed by Cecil et al. (1988) in Large White Turkeys ( sperm per male), an indication that reproductive parameters may vary greatly between lines of turkey males of various genetic origins. From a comparative standpoint, our results on DSO values in males collected five to seven times weekly are similar to those previously observed in muscovy ducks (Tan, 1980) and in fowl males subjected to comparable semen collection

5 1038 NOIRAULT AND BRILLARD TABLE 3. Sperm viability percentages, daily sperm output (DSO), and DSO of viable spermatozoa in turkey males (n = 7 per group) submitted to various frequencies of semen collection (x ± SE) Frequency Viability DSO Viable DSO (%) ( 10 9 ) ( 10 9 ) Once every 2 wk ± 0.54 d 0.27 ± 0.03 e 0.23 ± 0.02 e Once per week ± 0.63 c 0.55 ± 0.06 d 0.47 ± 0.02 d Twice per week ± 0.40 c 1.02 ± 0.08 c 0.90 ± 0.05 c Three times per week ± 0.26 b 1.57 ± 0.09 b 1.38 ± 0.08 b Five times per week ± 0.33 a 1.83 ± 0.08 a 1.59 ± 0.08 a a emeans within a column with no common superscript differ significantly (P< 0.05). 1Four consecutive weeks. 2Two consecutive weeks. frequencies (de Reviers and Williams, 1981). Because under practical field conditions a minimum amount of 150 to sperm per hen are required to inseminate turkey hens at weekly intervals (Van Wambeke and Huyghebaert, 1989), approximately 50 to 75 turkey hens could hypothetically be inseminated weekly from each male undergoing five to seven semen collections per week compared to about 20 to 30 hens per week if males are subjected to only one collection once per week. In the present experiment, a dual fluorescence test (Sybr14-PI) was used to quantify the viability/ nonviability of spermatozoa present in ejaculates of turkey males submitted to various semen collection frequencies (Donoghue et al., 1995). Our results in Group 2 (collected once per week) fully confirm earlier findings of Donoghue et al. (1995) regarding the mean percentage of sperm viability observed in turkeys subjected to the same rhythm of semen collection. However, in the present experiment we also observed a persistent, although moderate, increase in the percentage of viable spermatozoa from males collected more frequently. Similarly, in males collected daily for 5 consecutive d/ wk, ejaculates obtained during the days immediately following a rest period contained higher percentages of nonviable spermatozoa than ejaculates collected during the last days of a given week. Although it remains to be determined in turkeys, the duration of spermatogenesis is constant within a given species (ram, Courot, 1962; bull, Attal and Courot, 1963; cockerel, de Reviers, 1968; duck, Marchand et al., 1977); leading to the conclusion that sperm viability in turkeys is at least partly influenced by the duration of in vivo sperm storage in the vas deferens. Hypothetically, aging spermatozoa may progressively lose membrane integrity due to peroxidation occurring in the male genital tract. Although not observed in turkey spermatozoa kept in vitro for limited periods of time (Cecil and Bakst, 1993), such damage has already been reported in fowl (Fujihara and Howarth, 1978; Wishart, 1984; Suraï et al., 1997). None of these studies could demonstrate a direct or indirect link between the rate of sperm viability and the duration of the storage in the male ducti deferens. However, previous studies have demonstrated a direct link between the frequency of semen collection and the subsequent fertilizing capacities of ejaculates (turkey, McCartney et al., 1958; fowl, McDaniel and Sexton, 1977). The present study therefore provides additional evidence for Brown s observations (1968) that the viability of spermatozoa is influenced by the frequency at which males are collected. It can therefore be concluded that varying the frequency of semen collection in male turkeys not only increases or impairs the quantities of spermatozoa TABLE 4. Percentage viability of spermatozoa in ejaculates collected from turkey males submitted to various frequencies of semen collection (x ± SE) Sperm viability by day of semen collection Frequency Monday Tuesday Wednesday Thursday Friday Once every 2 wk ± Once per week ± Twice per week ± 0.47 a ± 0.52 a... Three times per week ± 0.47 a ± 0.56 a ± 0.58 a Five times per week ± 0.32 c ± 0.47 c ± 0.32 b ± 0.55 b ± 0.41 a a cwithin a given row, means with no common superscript differ significantly (P < 0.05). 1Four consecutive weeks. 2Two consecutive weeks. (%)

6 SEMEN OUTPUT IN MALE TURKEYS 1039 available for insemination (with further consequences on the number of females to be inseminated per male), but also modifies the initial rate of sperm viability in ejaculates, with ultimately the risk of disturbing the overall reproductive performance in breeder flocks. At a strategic level, the practical use of adequate frequencies of semen collections, coupled with the Sperm Motility Test recently developed by Holsberger et al. (1998), should provide useful tools for optimizing the fertilizing potential of the best sires in both selected lines and commercial situations. REFERENCES Amann, R. P., Sperm production rates. Pages in: The Testis. Vol. 1. A. D. Johnson, W. R. Gomes, and N. L. Vandemark, ed. Academic Press, New York, NY. Attal, J., and M. Courot, Développement testiculaire et établissement de la spermatogénèse chez le taureau. Ann. Biol. Anim. Biochem. Biophys. 3(3): Bakst, M. R., and H. C. Cecil, 1983a. Gross appearance of turkey cloacae before and after single or multiple manual semen collections. Poultry Sci. 62: Bakst, M. R., and H. C. Cecil, 1983b. Histology of turkey papillae after manual semen collection. Poultry Sci. 62: Brillard, J. P., and M. de Reviers, Testis development and daily sperm output in guineas submitted to progressively increasing daily photoperiods at different ages. Poultry Sci. 64: Brown, K. I., Efficient use of turkey males. Technical Bulletin, Ohio Agricultural Research and Experimental Station; Res. Sum. 33:5 7. Burrows, W. H., and J. P. Quinn, The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: Cecil, H. C., Effects of frequency of semen collection on reproductive performance of male turkeys fed low protein diets during the breeder period. Poultry Sci. 61: Cecil, H. C., M. R. Bakst, and A. Monsi, Daily output of spermatozoa and extragonadal spermatozoal reserves in turkeys. Poultry Sci. 67: Cecil, H. C., and M. R. Bakst, In vitro lipid peroxidation of turkey spermatozoa. Poultry Sci. 72: Chalah, T., and J. P. Brillard, Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (Sybr14/IP). Theriogenology 50(3): Courot, M., Développement du testicule chez l agneau. Etablissement de la spermatogénèse. Ann. Biol. Anim. Bioch. Biophys. 2(1): De Reviers, M., Détermination de la durée des processus spermatogénétiques chez le coq a l aide de thymidine tritiée. VI Cong. Int. Anim. Insem. Artif. Paris 1: De Reviers, M., Évaluation de la production de spermatozoïdes chez le coq. Ann. Biol. Anim. Biochem. Biophys. 12(1): De Reviers, M., and J. Williams, Predicting the adult daily sperm output after the first ejaculates in cokerels raised under different photoschedules. Reprod. Nutr. Dev. 21(6B): Donoghue, A. M., D. L. Garner, D. J. Donoghue, and L. A. Johnson, Viability assessment of turkey sperm using fluorescent staining and flow cytometry. Poultry Sci. 74: Fujihara, N., and B. Howarth, Lipid peroxidation in fowl spermatozoa. Poultry Sci. 57: Holsberger, D. R., A. M. Donoghue, D. P. Froman, and M. A. Ottinger, Assessment of ejaculate quality and sperm characteristics in turkeys: sperm mobility phenotype is independent of time. Poultry Sci. 77: Lake, P. E., and O. Ravie, Effect on fertility of storing turkey semen for 24 hours at 10 C in fluids of different ph. Br. Poult. Sci. 32: Lorenz, F. W., N. E. Wilson, and V. S. Asmundson, Relation of frequency of collection to amount of semen obtained from turkey males. Poultry Sci. 34: Marchand, C. R., L. Gomot, and M. de Reviers, 1977 Etude par radiographie et marquage à la thymidine tritiée de la durée de la spermatogénèse du canard de barbarie (Carina moschata L.). C. R. Séances Soc. Biol. 171(4): McCartney, M. G., R. D. Chamberlin, R. D. Carter, and J. W. Wyne, Effect of frequency of semen collection on fertility, hatchability and spermatozoa concentration in the turkey. Poultry Sci. 37: McDaniel, G. R., and T. J. Sexton, Frequency of semen collection in relation to semen volume, sperm concentration and fertility in the chicken. Poultry Sci. 56: Suraï P. F., E. Kutz, G. J. Wishart, R. C. Noble, and B. K. Speake, The relationship between the dietary provision of a-tocopherol and the concentration of this vitamin in the semen of chicken:effects on lipid composition and susceptibility to peroxidation. J. Reprod. Fertil. 110: Tan, N. S., The frequency of collection and semen production in muscovy ducks. Br. Poult. Sci. 21: Van Wambeke, F., and G. Huyghebaert, Current role of semen storage and artificial insemination in the turkey industry. Br. Poult. Sci. 30: Wishart, G. J., Effects of lipid peroxide formation in fowl semen on sperm motility, ATP content and fertilizing ability. J. Reprod. Fertil. 71: