Daily Output of Spermatozoa and Extragonadal Spermatozoal Reserves in Turkeys

Daily Output of Spermatozoa and Extragonadal Spermatozoal Reserves in Turkeys HELENE C. CECIL and MURRAY R. BAKST US Department of Agriculture, Agricultural Research Service, Avian Physiology Laboratory, Beltsville, Maryland 20705 ALEXIS MONSI Department of Animal Science/Food Science and Technology, Rivers State University of Science and Technology, Port Harcourt, Nigeria

ABSTRACT Daily spermatozoal output (DSO) was estimated by determining the total number of spermatozoa in ejaculates of male line breeder turkeys ejaculated daily for 15 days. The DSO was constant at 520 million spermatozoa after the first 6 days of semen collection. Testicular spermatozoal reserves (TSR) and extragonadal sperm reserves (EGR) were measured 24 h after the last semen collection from turkeys ejaculated 1 x/day for 15 consecutive days (ejaculated) and 21 days after the last semen collection from turkeys previously ejaculated 1 x /wk for 12 wk (rested). The TSR concentrations were similar (P>.05) for ejaculated and rested groups, respectively: 117 x 106 and 119 x 106/g for right testis; 127 x 106 and 135 x 106/g for left testis. The total TSR were also similar (P>.05): 3,308 x 106 (ejaculated) and 4,343 x 106 (rested). However, EGR for ejaculated and rested groups were significantly different, with the following values, respectively: ductus deferens, 3,160 x 106 and 10,320 x 106 (P = .0005); epididymis, 58 x 106 and 204 x 106 (P<.004); total EGR, 3,248 x 106 and 10,524 x 106 (P<.005). This study shows that although TSR was not affected by daily semen collection, EGR was depleted by 70% of the rested value (P<.005) when the turkeys were ejaculated daily for 15 days. (Key words: ejaculate, spermatogenesis, testes, semen production, turkey) 1988 Poultry Science 6 7 : 3 2 7 - 3 3 2 INTRODUCTION

Semen volume, spermatozoal concentration, and spermatozoal viability are routinely measured to assure that the best quality semen is used for artificial insemination of turkeys. However, little is known of the quantitative aspects of the physiological mechanisms that control and influence ejaculation and semen volume. Three factors that could possibly influence the volume of ejaculates and their spermatozoal concentrations are: 1) testicular spermatozoal reserves (TSR), the number of mature spermatids and spermatozoa in the testes; 2) daily spermatozoal output (DSO), the number of spermatozoa released each day from the testes to the epididymides and ductus (d.) deferens; and 3) extragonadal spermatozoal reserves (EGR), the number of spermatozoa contained in the epididymides and d. deferens. Estimation of TSR has been used as a method to assess the production rate of spermatozoa in chicken (Verma et al., 1966; de Reviers, 1972; Blazak and Fechheimer, 1981) and turkey testes (Bakst and Cecil, 1984). Also, the total number 111

of spermatozoa in ejaculates, expressed on a daily basis, has been used to estimate DSO from the chicken (de Reviers and Williams, 1981) and guinea fowl testes (Brillard and de Reviers, 1981; 1985). The DSO is an important factor when semen is frequently collected because the number of ejaculated spermatozoa then approaches the DSO. However, because the d. deferens stores spermatozoa and only a portion of these are ejaculated (Cecil and Bakst, 1984), EGR directly affects the number of spermatozoa in the ejaculate. The EGR has been measured in the chicken (de Reviers, 1972; 1975), turkey (Bakst and Cecil, 1984; Cecil and Bakst, 1984) and Japanese quail (Clulow and Jones, 1984). The present study was conducted to determine the DSO by daily ejaculation and to compare the TSR and EGR of turkeys ejaculated daily with turkeys that had been sexually rested. MATERIALS AND METHODS

Large White male breeder turkeys raised in floor pens were used in this study. The ad libitum

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(Received for publication February 9, 1987)

328

CECIL ET AL.

NY). Homogenization resulted in the disruption of all cells but left nuclei intact. Duplicate aliquots of the homogenate were diluted 1:1 with embalming fluid (Vita-firm Index 24, Norris A. Dodson, Inc., Washington, DC). Only fully condensed heads of spermatids and spermatozoa, which were differentiated from other germ and somatic cell nuclei by their density, curvature and length, were counted. Condensed heads were counted using a hemocytometer under a phase contrast microscope at 400X magnification. Semen from each isolated d. deferens

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FIGURE 1. Semen volume, spermatozoal concentration. and total number of spermatozoa in ejaculates collected on 15 successive days. Analyses of the data to determine the equation of the line which could best fit the data gave the following equations: volume (R2 = .33); log(mL) = -.431 - 1.29(log day) + .88 flog day)2; concentration (R2 = .38); log(billion sperm per mL) = .924 - .395(log day); total sperm (R2 = .46); log(billion sperm) = .390-.128(log day) + .57(log day)2.

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dietary regimen was: corn-soybean meal diet containing 30% protein (0 to 4 wk), 26% protein (4 to 8 wk), 19% protein (8 to 12 wk), 17% protein (12 to 17 wk), and a corn diet containing 8% protein from 17 wk to the end of the experiment at 45 wk. Dietary compositions are as described in Cecil (1984). The lighting regimen was: 24 h light for the first 2 days, then a daily step down of 2 h/day until 14 h light/day at 7 days; 14 h light/day until 10 wk; 12 h of 54-lx incandescent light from 10 to 26 wk; 14 h of 6.5-lx incandescent light from 26 wk to the end of the experiment. Turkeys weighed 15.4 and 18.3 kg at 28 and 45 wk of age, respectively. Once a week from 31 to 42 wk of age, semen was collected from 65 males using two cloacal strokes (Bakst and Cecil, 1983).The semen was collected into a conical graduated centrifuge tube and the volume was measured. The concentration of spermatozoa in each semen sample was measured spectrophometrically in a Klett Summerson colorimeter (Klett Mfg. Co., Inc., New York, NY) after diluting 10 u-L semen with 10 mL of 3% NaCl (Cecil, 1982). Total sperm per ejaculate reached a plateau at 35 wk of age. At 42 wk of age, 10 pairs of turkeys were selected based on the total number of spermatozoa per ejaculate for the previous 5 wk (range equals 2.0 to 3.3 x 109 spermatozoa per ejaculate); turkeys in each pair were matched for equal numbers of spermatozoa in their semen samples. One turkey of each pair was sexually rested for 7 days and then semen was collected with three to four cloacal strokes each day for 15 consecutive days (ejaculated). It has been determined that after two strokes the number of spermatozoa ejaculated is markedly reduced and that three to four strokes ensure a complete collection (Cecil and Bakst, 1985). The second turkey from each pair was not ejaculated for 21 days (rested). Turkeys were killed by an overdose of pentobarbital 24 h after the last semen collection (ejaculated) or after 21 days of sexual rest (rested). The testes, excurrent duct system, and cloaca were removed in toto. The epididymal region was isolated from the testes, leaving the testicular capsule intact. The proximal end of the d. deferens was clamped, its distal end isolated from the cloaca, and the papillae clamped. Each testis and epididymis was weighed, minced, diluted with 15 vol of .9% NaCl containing .05% Triton-X 100, and homogenized 30 s (Blazak and Fechheimer, 1981) with a Polytron (Brinkmann Instruments, Westbury,

SPERMATOZOA RESERVES IN TURKEYS

RESULTS AND DISCUSSION

Four of the 10 turkeys in the ejaculated group had wide fluctuations in their day-to-day ejaculate volume and total number of spermatozoa per ejaculate and did not reach a predictable plateau with daily semen collections. Consequently, these four turkeys were not used to predict the DSO. However, six of the ten turkeys in the ejaculated group maintained relatively constant semen volume (Figure 1A) and total

number of spermatozoa per ejaculate (Figure IC) after 6 days of semen collection; only data for these six and their pair mates are reported. After 7 days of sexual rest, the Day 1 ejaculate contained 2,530 X 106 spermatozoa. On successive days, semen volume and sperm concentration (Figure IB) declined. On the 4th day of semen collection a level of 520 x 106 spermatozoa/ejaculate was reached and maintained for the remaining semen collections. Therefore, the DSO for the turkey is defined as 520 x 106 spermatozoa and is about one-fourth that of the chicken (2,000 X 106;deReviers and Williams, 1981), and eight times that of the guinea fowl (60 to 80 x 10 6 ;BrillardanddeReviers, 1981; 1985). Why such a wide variation in DSO exists between these galliformes is not known. Testicular weights and spermatozoal and extra gonadal reserves from turkeys after daily ejaculation for 15 days or after 21 days of sexual rest are shown in Tables 1 and 2. Statistical analyses showed no side x treatment interactions (Tables 1 and 3). However, the left testis weighed more (P = .0008) and had a greater concentration of spermatozoa (P = .009) and a greater total number of spermatozoa (P = .0006) than the right testis (Table 1). The left epididymis also weighed more (P = .005) and had a greater total number of spermatozoa (P = .03) than the right epididymis (Tables 2 and 3). Although the left testis and left epididymis had a greater number of spermatozoa than the right, there were no differences between the left

TABLE 1. Testicular weights and spermatozoal reserves from turkeys after daily ejaculation for 15 days (ejaculated) or after 21 days of sexual rest (rested) and analysis of variance Testis weight Treatment

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was manually expressed into a tube and volume and spermatozoal concentration determined as described for ejaculates. Testicular and epididymal weights, spermatozoa per gram testis, spermatozoa per testis, spermatozoa per gram epididymis, total spermatozoa per epididymis, and the volume, spermatozoal concentration, and total spermatozoa in right and left d. deferens semen of ejaculated and rested groups were compared by analysis of variance using the General Linear Model Procedure, type III SS (SAS Institute Inc., 1985). Experimental data were analyzed as a completely randomized split plot with six replicates (birds) per treatment. Treatments (ejaculated vs. rested groups) were whole plot; sides (right vs. left) were subplot treatments. Sources of variation were: treatment, error a, side, treatment x side, and error b. Treatment effect was ejaculated vs. rested group; side effect was right vs. left.

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and right ductus deferens in number of spermatozoa (Tables 2 and 3). The weight, spermatozoal concentration, and total spermatozoa content of the testes were similar (P>.05) for the ejaculated and rested groups (Table 1), indicating no effect of frequency of semen collection on testicular spermatozoa production. If one assumes a constant rate of replenishment of spermatozoa in the testes, then with a DSO of 520 x 106, a TSR of 3,308 x 106 (ejaculated total spermatozoa, Table 1) represents a 6-day reservoir of maturing spermatozoa. Because 1) only condensed heads of spermatids and spermatozoa were counted and 2) it is assumed that the time required for spermatids to become transformed into spermatozoa is a constant, then a TSRrDSO ratio of 6 could be interpreted to mean that late spermiogenesis requires 6 days. Following the time course of exogenous thymidine incorporation into meiotic germ cells, de Reviers (1968) estimated spermiogenesis to take 7 days in the chicken. The d. deferens semen volume and total spermatozoa of the rested group were 2.8 and 3.3 times greater (P<.0006), respectively, than that of the ejaculated group (Tables 2 and 3). The spermatozoal concentration of the d. deferens semen in the rested group was also greater (P = .033) than that of the ejaculated group. The epididymal weights were 1.3 times (P = .04), epididymal spermatozoa per gram were 3.3 times (P = .006), and epididymal total spermatozoa were 3.5 times (P = .004) greater in the rested group than in the ejaculated group. The total EGR (d. deferens + epididymides) was 10,524 X 106 in the rested group, equivalent to 20 days DSO, and 3,218 x 106 in the ejaculated group, equivalent to 6 days DSO. The EGR of the rested group (10,524 X 106) is assumed to represent the maximum storage capacity of the excurrent duct system of these turkeys. With a once-a-week collection, the ejaculate of the rested group contained 2,800 x 106 sperm or 29% of the EGR, whereas with daily collections the ejaculate contained 520 X 106 spermatozoa or 16% of the 3,218 X 106 spermatozoa in the EGR of the ejaculated group. Although statistical comparison shows that a larger proportion of the EGR was ejaculated in the rested group than the ejaculated group (29% vs. 16%, P<.05), from a practical standpoint the difference between the two groups is small and illustrates that the abdominal massage method stimulates the movement of approxi-

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CECIL ET AL. male Japanese quail, Coturnix coturnix. J. Reprod. Fert. 64:259-266. de Reviers, M., 1968. Determination de la duree des processus spermatogenetique chez le coq a la aide de la thymidine tritiee. Proc. 6th Int. Congr. Anim. Reprod. Artif. Insem., Vol. 1:183-185. de Reviers, M., 1972. Evaluation des reserves spermatiques dans les voies deferentes du coq. Ann. Biol. Anim. Biochim. Biophys. 12:5-11. de Reviers, M., 1975. Sperm transport and survival in male birds. Pages 10-16 in: The Biology of Spermatozoa. INSEM Int. Symp. Nouzilly, 1973. Kargel, Basel, Switzerland. de Reviers, M., and J. Williams, 1981. Predicting the adult daily sperm output after the first ejaculates in cockerels raised under different photoschedules. Reprod. Nutr. Dev. 21:1113-1124. SAS Institute Inc.. 1985. SAS/STAT Guide for Personal Computers, Version 6 Ed. SAS Institute Inc., Cary, NC. Verma, M. C , V. D. Sharma, andG. Singh, 1966. Studies on sperm production. III. Testicular and epididymal sperm reserves in small animals (rabbit, guinea-pig, albino rat and fowl). Indian J. Vet. Sci. Anim. Husb. 36:109-118.

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Brillard, J. P., andM. de Reviers, 1981. Testis development and daily sperm output in guinea fowl raised under constant daily photoperiods. Reprod. Nutr. Dev. 21:1105-1112. Brillard, J. P., andM.deReviers, 1985. Testis development and daily sperm output in guineas submitted to progressively increasing daily photoperiods at different ages. Poultry Sci. 64:713-717. Cecil, H. C , 1982. Effects of frequency of semen collection on reproductive performance of male turkeys fed low protein diets during the breeder period. Poultry Sci. 61:1866-1872. Cecil, H. C , 1984. Effect of dietary protein and light restriction on body weight and semen production of breeder male turkeys. Poultry Sci. 63:1175-1183. Cecil, H. C , and M. R. Bakst, 1984. Testicular weights, ductus deferens semen volumes, and sperm concentration of turkeys with high and low ejaculate volumes. Poultry Sci. 63:1432-1437. Cecil, H. C , and M. R. Bakst, 1985. Volume, sperm concentration, and fertilizing capacity of turkey ejaculates obtained from successive cloacal strokes during semen collection. Poultry Sci. 64:1219-1222. Clulow, J., and R. C. Jones, 1984. Production, transport, maturation, storage and survival of spermatozoa in the