Semen Production by the Emu (Dromaius novaehollandiae). 2. Effect of Collection Frequency on the Production of Semen and Spermatozoa

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1 Semen Production by the Emu (Dromaius novaehollandiae). 2. Effect of Collection Frequency on the Production of Semen and Spermatozoa I. A. MALECKI,*, G. B. MARTIN,*, and D. R. LINDSAY* *Animal Science, Faculty of Agriculture, The University of Western Australia, Nedlands, W A 6009, Australia, and CSIRO Division of Animal Production, Private Bag, PO Wembley, W A 6014, Australia ABSTRACT This experiment tested the hypothesis that frequent collection of semen from emus would increase the total output of semen and spermatozoa over less frequent collection. Semen was collected from trained male emus using an artificial cloaca. In Experiment 1, semen was collected from males every 4th d (96- h interval), every 2nd d (48-h interval), and every day (24-h interval) for 16 d. In Experiment 2, semen was collected daily (24-h interval), twice daily (6-h interval), Received for publication May 24, Accepted for publication December 16, and three times per day (3-h interval) over 6 d. Twicedaily collections yielded twice as much volume and number of spermatozoa than daily collections. Collecting semen three times per day did not yield extra semen, as it adversely affected the libido of males. Twice-daily collections appears to yield the optimum output over the 6 d of this experiment, but longer periods of collection need to be studied. (Key words: emu, ratite, spermatozoa, semen collection, artificial cloaca) INTRODUCTION Introduction of artificial insemination into the emu industry would permit a greater rate of genetic improvement, reduce the number of males needed for breeding, and increase output of birds from the farm. Other avian industries have benefited from artificial insemination by developing breeding lines of desirable traits or using semen of superior males to increase production. The success of this approach will not only depend on our ability to develop systems for semen collection, but also upon the rate of sperm production and the availability of spermatozoa for artificial insemination. To obtain the maximum number of spermatozoa and maintain high rates of sperm production, the semen needs to be collected at a frequency that maintains output over time. In birds, the optimal frequency differs among species and even among breeds of the same species. In broiler and layer chickens, collecting semen three times per week yielded the best semen and sperm output per ejaculate in one study (McDaniel and Sexton, 1977), but in another, Fuquay and Renden (1980) reported that collections five times per week gave the highest total number of sperm per ejaculate. In both studies, total weekly output was higher with collections 5 times per week than with 3 times per week, but even more semen was gained from collecting 10 times per week. Even though high frequency collections appeared to be 1997 Poultry Science 76: feasible for several weeks, they had an adverse effect on the males and output of spermatozoa. In Muscovy drakes, daily ejaculations can be carried out (Tan, 1980). Even less frequent collections are recommended for turkey males because daily collections yield a high output but the volume gradually decreases during the 1st wk (Lorenz et al., 1955; McCartney et al., 1958). As turkeys require about 3 d to replenish their sperm reserves following each collection (Nestor and Brown, 1971; Bakst and Cecil, 1981), collecting semen every other day (three times per week) has been recommended (Cooper, 1977). No information on semen production in the emu has been published, so our first step was to determine the optimum frequency of collection. We hypothesized that the emu would be constrained in the same way as the other birds, so that there would be a frequency above which there would be no significant gains in semen yield. A preliminary report of this study has been presented by Malecki et al. (1994). MATERIALS AND METHODS Birds and Management The experiments were conducted in 1993 and 1994 at the Medina Research Station of the Agriculture Western Australia, near Perth (32 S). For Experiment 1, 12 mature male emus (aged 2 to 5 yr) were selected from a group of 16 that were trained to ejaculate into an artificial cloaca using two different methods: one using a female teaser, the other without a 622

2 teaser, in which the male mounted the collector (Malecki et al., 1996). The males were penned individually next to each other in 3 20 m pens arranged in two parallel rows of 12. The remaining pens contained 12 females that were to be used as teasers. The birds consumed water and feed ad libitum. A breeder emu ration was formulated at the Station (O Malley, 1996). For Experiment 2, which was done the following year, 12 males were selected, including 8 from Experiment 1 and 4 new males that had been successfully trained to ejaculate into an artificial cloaca using a teaser female. The eight birds from the previous experiment underwent retraining. PRODUCTION OF SEMEN AND SPERMATOZOA IN EMU 623 Experiment 1 Five males that had been trained for collection using teasers, and four trained without teasers were chosen for the experiment and randomly divided into three groups of three. They were subjected to three collection frequencies: once daily, once every 2nd d, and once every 4th d, over a collection period of 16 d, using a triple Latin square design that allowed each male to be tested with each frequency of collection. All males were ejaculated once a day before the experiment started and 1 d was allowed between each experimental period during which semen was collected once from all males. Collection commenced at 0830 h each day. One male stopped producing sperm at the beginning of the second experimental period and its data were excluded from the analysis. Experiment 2 Nine males (seven using teasers and two without teasers) were divided into three groups of three and subjected to collection frequencies of once daily (0900 h), twice daily (0900 and 1500 h), or three times per day (0900, 1200, and 1500 h) for 6 d in the same Latin square design as in Experiment 1. As in Experiment 1, all males were ejaculated once a day before the experiment started and 1 d between 6-d test periods was allowed when semen was collected only once from all birds. Measurement of Male Libido In Experiment 2, we anticipated a decreased willingness of males to ejaculate due to increased frequency of collection, which is a feature of the artificial vagina method of collection in mammals (Salamon and Lindsay, 1961; MacMillan, 1980). Therefore, the libido was gauged from the reaction time, as measured by the delay (in seconds) from the moment that the male entered the pen of a female to the time of ejaculation. Where collections were made without a female, the delay was measured from the moment that the collector entered the pen until the emu ejaculated into the artificial cloaca. Only days with a complete number of ejaculations were included in the analysis to avoid the potential bias of reaction times to a refused ejaculation. FIGURE 1. Effect of the collection frequency on total output of semen ( ) and spermatozoa ( ) over the collection period in Experiments 1 (upper panel, n = 8) and 2 (lower panel, n = 9, Mean ± SEM). Means with different superscript differ significantly (P < 0.05). Semen Measurements and Analysis of Data Volume of semen was measured with a graduated pipette and the spermatozoa were counted with a hemocytometer after dilution 1:400 with saline and ethanol (1% vol/vol). Two hemocytometer counts (a total of four replicates) for each sample were made. All data were subjected to ANOVA using Super- ANOVA software, (Abacus Concepts, 1989). The differences between means were tested by Fisher s Protected LSD. The total daily output for each frequency of collection was calculated by dividing the total volume of semen and number of spermatozoa collected in a test period by the number of days in that period. The relationship between semen characteristics and day of collection was examined by regression analysis. Data are presented as mean ± SEM and P < 0.05 is considered significant. RESULTS Total Output of Semen and Spermatozoa Main Effects In Experiment 1, the frequency of collection significantly affected the total volume (P < 0.01) and total number of spermatozoa (P < 0.05) produced over the 16-d collection period. The greatest semen volume and number of spermatozoa were obtained from daily collections (Figure 1). For both variables, the values differed significantly between collections made daily and every 2nd d. The values for collections every 2nd d were double those for collection every 4th d but the differences were not significant.

3 624 MALECKI ET AL. TABLE 1. Effect of twice-daily collections on the ejaculate characteristics in collections made 6 h apart in Experiment 2 1 Character Collection 1 Collection 2 n = 49 n = 40 Volume, ml 0.7 ± ± 0.1 Number of sperm, ± ± 0.4 Concentration, 10 9 /ml 3.3 ± ± 0.2 1All values are means ± SEM. Means between Collection 1 and 2 did not differ significantly (P > 0.05). In Experiment 2, there was also a significant effect of the frequency of collection on the total volume (P < 0.01) and total number of spermatozoa (P < 0.01). Twice-daily collections yielded significantly more semen and spermatozoa than daily collections, but there was no further increase with collections three times per day. The linear increase in the total volume and number of spermatozoa appeared to become asymptotic around twice-daily (Figure 1). The data from twice-daily collections were analyzed for differences in the total output and mean per ejaculate between the first and second collections taken 6 h apart. The total semen volume and number of spermatozoa did not differ between collections obtained on successive days of the period. The mean ejaculate volume, concentration, and number of spermatozoa were also similar in the two ejaculates (Table 1). In both experiments, there was a significant betweenmale effect (P < 0.01) on the total volume of semen and the total number of spermatozoa, but semen yields did not differ between periods of collection (Table 2). Semen Characteristics Within experiments, the mean volume, concentration, and number of spermatozoa per ejaculate did not differ between collection frequencies. Over both experiments, the mean ejaculate had a volume of 0.61 ± 0.06 ml (range 0.27 to 1.39), and contained 1.94 ± sperm (0.71 to 4.75) at a concentration of 3.34 ± sperm/ml (1.81 to 4.67). Daily Output of Semen and Spermatozoa The lowest daily outputs were observed when birds were ejaculated once every fourth day (Table 3). A significant increase in the daily output per male was gained as the frequency increased to two collections per day. Periods of Collection and Entire Experiments Over the 16-d period of collection in Experiment 1, the volume of semen and the concentration and number of spermatozoa did not change. Over the entire duration of the experiment there was a gradual decline in the average volume of the ejaculate (P < 0.05), whereas the concentration and number of spermatozoa were not affected. Over the 6-d period of daily collections in Experiment 2, there was no change in the semen volume, or in the concentration and number of spermatozoa on successive days (Figure 2). With twice daily and three times daily collections, the total volume and total number of spermatozoa declined initially and then the output appeared to stabilize around Days 3 and 4. Over the 20 d of the entire experiment, no diminution in any semen characteristic was observed. Effect of Higher Frequencies of Collection on Male Behavior and Output of Semen In Experiment 2, collection frequency affected the number of ejaculates that was collected. In 324 attempts, a total of 246 ejaculates were successfully collected. The rate of success was 85.2% with daily and 83.3% with twicedaily collections. The number of successful collections at three times daily was 85% on Day 1 but it then decreased to 60% on Day 2 and remained at this level for the rest of the study, so the overall rate of success was only 67.9%. The frequency of collection significantly affected the reaction time of males (P < 0.01). The mean reaction time TABLE 2. Mean output of semen and spermatozoa in the emu (Dromaius novaehollandiae) in Experiments 1 and 2 Experiment 1 Experiment 2 Variable Volume No. of sperm Volume No. of sperm (ml) ( 10 9 ) (ml) ( 10 9 ) Male ± ± ± ± 2.1 Range 0.5 to to to to 52.3 CV, % Period ± ± ± ± ± ± ± ± ± ± ± ± 4.0 1Values are means ± SEM for the period of 16 d in Experiment 1 and 6 d in Experiment 2. Effect of male in each experiment was significant (P < 0.01). 2Means ± SEM between periods of collection within each experiment were not different (P > 0.05).

4 PRODUCTION OF SEMEN AND SPERMATOZOA IN EMU 625 did not differ between twice-daily and daily collections, but the males took significantly more time to ejaculate (P < 0.05) when their semen was collected three times per day (Figure 3). DISCUSSION In the male emu, the highest output of semen volume and number of spermatozoa is obtained using twicedaily collections. This frequency did not affect the mating behavior of the males, as indicated by failure rate for collection and reaction time. Attempting to collect semen three times per day did not yield more semen, as the frequency reduced the libido of males and increased the rate of failure. With twice-daily collections, the yield of semen did not change over the 6-d test period and there was no difference between successive collections made 6 h apart in the total output and per ejaculate characteristics. Emus thus appear to be more consistent than broiler chickens (McDaniel and Sexton, 1977) or ducks (Tan, 1980) ejaculated twice-daily. However, in both of these studies, shorter intervals (5 and 2 h, respectively) between two collections were used. We could expect that the first ejaculate would contain a greater proportion of the reserves of spermatozoa, which are located in the distal halves of the ducti deferentes (de Reviers, 1975; Lake, 1981). Depending on the rate of replenishment, time is needed to refill the ducti with semen (Bakst and Cecil, 1981). This conclusion is supported by the work of McDaniel and Sexton (1977), who collected significantly less spermatozoa in the second ejaculate after a 3-h interval than after a 5-h collection interval. With twice-daily collections in our emus, the volume of the ejaculate and the output of spermatozoa stabilized, suggesting that by this time a balance between the daily output and production of spermatozoa was reached. This result suggests that daily collections will not fully utilize the total daily production of spermatozoa by the testes and some spermatozoa might be lost. The decline in the average volume of the ejaculate over the 50 d of Experiment 1 appears to be a seasonal effect rather than a consequence of the collection procedure. In Western Australia, emus breed from March until September (Williams et al., 1995). In the male, testicular size and plasma testosterone concentration change seasonally and sperm production terminates around September-October (I. Malecki, unpublished data). The experiment was carried out in the second half of the breeding season. Throughout the breeding season, due to the strong positive correlation between daily sperm production and testicular size (de Reviers and FIGURE 2. Changes in the total output of semen (upper panel) and number of spermatozoa (lower panel) during 6 d test in Experiment 2 (n = 9). Points are Mean ± SEM. Frequency of collection: every 3 h; ÿ every 6 h; o every 24 h. FIGURE 3. Effect of the frequency of collection on reaction time in Experiment 2 (Mean ± SEM, n = 9). Means with no common letter differ significantly (P < 0.05).

5 626 MALECKI ET AL. TABLE 3. Effect of collection frequency on daily output of semen and spermatozoa in the emu (Dromaius novaehollandiae) 1 Collection interval Semen volume Number of spermatozoa Mean ± SEM Range CV Mean ± SEM Range CV (h) (ml/d) (%) ( 10 9 /d) (%) ± to ± to ± to ± to ± to ± to ± to ± to ± to ± to ± to ± to Calculated from data presented in Figure 1. Williams, 1984), the semen yield from the male emu will be affected by the seasonal changes in the testes. In the chicken it was feasible, although with an adverse effect on the males, to ejaculate males 10 times per week for up to 15 wk (McDaniel and Sexton, 1977; Fuquay and Renden, 1980). The length of the time that male emus could sustain a twice daily rate remains to be studied. Individual males vary greatly in semen yields and ejaculate characteristics and we can distinguish low and high sperm-producing male emus; therefore, some males can be used more intensively than the mean of those used in our studies. REFERENCES Abacus Concepts, SuperANOVA. Abacus Concepts, Inc., Berkeley, CA. Bakst, M. R., and H. C. Cecil, Changes in the characteristics of turkey ejaculated semen and ductus deferens semen with repeated ejaculations. Reprod. Nutr. Dev. 21: Cooper, D. M., Artificial insemination. Pages in: Poultry Diseases. R. F. Gordon, ed. Baillere Tindall, London, UK. de Reviers, M., Sperm transport and survival in male birds. Pages in: The Biology of Spermatozoa. E.S.E. Hafez and C. G. Thibault, ed. Karger, Basel, Switzerland. de Reviers, M., and J. B. Williams, Testis development and production of spermatozoa in the cockerel (Gallus domesticus). Pages in: Reproductive Biology of Poultry. F. J. Cunningham, P. E. Lake, and D. Hewitt, ed. British Poultry Science, London, UK. Fuquay, J. I., and J. F. Renden, Reproductive performance of broiler breeders maintained in cages or on floors through 59 weeks of age. Poultry Sci. 59: Lake, P. E., Male genital organs. Pages 1 61 in: Form and Function in Birds. A. S. King, and J. McLelland, ed. Academic Press, New York, NY. 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: McCartney, M. G., V. 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: MacMillan, K. L., Significance of behavior in semen collection. Pages in: Behaviour in Relation to Reproduction, Management, and Welfare of Farm Animals. M. Wodzicka-Tomaszewska, T. N. Edey, and J. J. Lynch, ed. University of New England Press, Armidale, N.S.W., Australia. Malecki, I. A., G. B. Martin, and D. R. Lindsay, Effect of collection frequency on semen output in the male emu (Dromaius novaehollandiae). Proc. Austr. Soc. of Anim. Prod. 20:434. Malecki, I. A., G. B. Martin, and D. R. Lindsay, Semen production by the male emu (Dromaius novaehollandiae). 1. Methods for collection of semen. Poultry Sci. 76: 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: Nestor, K. E., and K. I. Brown, Semen production in turkeys. Poultry Sci. 50: O Malley, P. J., An estimate of the nutritional requirements of emus. Pages in: Proceedings of An International Conference held at Dalton-Ellis Hall: Improving our understanding of ratites in a farming environment March University of Manchester, Manchester, UK. Salamon, S., and D. R. Lindsay, The training of rams for artificial vagina, with some observations on ram behavior. Pages in: Proceedings of a Conference held at the School of Wool Technology Artificial Breeding of Sheep in Australia, University of New South Wales, 8 10 August 1961, E. M. Roberts, University, Sydney, NSW, Australia. Tan, N. S., The frequency of semen collection and semen production in Muscovy ducks. Br. Poult. Sci. 21: Williams, K. E., N. S. Tan, P. O Malley, M. A. Blackberry, P. J. Sharp, and G. B. Martin, Differences in serum concentrations of testosterone and prolactin in broody and non-broody male emus (Dromaius novaehollandiae). Page 111 in: Proceedings of the 27th Annual Conference of the Australian Society for Reproductive Biology, September 1995, Melbourne, Victoria, Australia.