Paternity Efficiency in Turkeys Differes Extensively After Hererospermic Insemination

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1 University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 1999 Paternity Efficiency in Turkeys Differes Extensively After Hererospermic Insemination Ann M. Donoghue USDA, annie@lpsi.barc.usda.gov Murray R. Bakst USDA Paul Drummond Tuskegee University Shakura Haqque Tuskegee University Edward J. Smith Tuskegee University See next page for additional authors Follow this and additional works at: Part of the Agricultural Science Commons Donoghue, Ann M.; Bakst, Murray R.; Drummond, Paul; Haqque, Shakura; Smith, Edward J.; and Donoghue, Daniel J., "Paternity Efficiency in Turkeys Differes Extensively After Hererospermic Insemination" (1999). Publications from USDA-ARS / UNL Faculty This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.

2 Authors Ann M. Donoghue, Murray R. Bakst, Paul Drummond, Shakura Haqque, Edward J. Smith, and Daniel J. Donoghue This article is available at of Nebraska - Lincoln:

3 01999 Applied Poultry Science, Inc PATERNITY EFFICIENCY IN TURKEYS DIFFERS EXTENSIVELY AFTER HETEROSPERMIC INSEMINATION ANN M. DONOGHUE' and MURRAY R. BAKST Germplasm and Gamete Physiology Laboratory, ART, USDA, Bldg. 200 (BARC-EAST), Beltsville, MD Phone: (301) FAX: (301) PAUL DRUMMOND, SHAKURA HAQQUE, and EDWARD J. SMITH School of Agriculture and Home Economics, Tuskegee University, Tuskegee, AL DANIEL J. DONOGHUE Center of Veterinary Medicine, FDA, Laurel, MD Primary Audience: Male Production Managers, Researchers DEsCRIPTroN OF Artificial insemination (AI), which is labor intensive, is used exclusively to produce the approhate1y300 Idion turkeys consumed in the U.S. Management practice dictates the utilization of heterospermic in- I ' 1 To whom correspondence should be addressed semination by pooling ejaculates from 10 to 15 toms [l]. In heterospermic insemination, the female receives sperm from more than one male so that a mixture Of sperm is availab1e for fertilization (see [2] for review). lbrkeysemen is Dooled Drimarflv for convenience. to reduce the effect; of poo; males, and to provide adequate semen for the large numbers of hens Journal of Applied Poultry Research 8 (1999), pp

4 Research Report DONOGHUE el al. 215 requiring AI on a weekly basis. It is assumed that all sperm from all toms are capable of transport and storage in the female tract and fertilizing eggs. However, paternity efficiency after pooling multiple ejaculates has not been previously determined in the turkey. Extensive semen analysis is fundamental to effective sire selection and reproductive management in livestock production systems where AI is practiced. However, sire selection is not a major consideration in the turkey industry, possibly due to the high numbers of males involved in weekly inseminations. Whereas certain male phenotypic traits of economic importance are more desirable than others, it has generally been assumed that differences in fertility between males were insignificant. The evaluation of individual males by laboratory personnel in the turkey industry is generally limited to assessment of semen color, ejaculate volume, and sperm motility and viability [3]. Scientists and farm personnel use several methods to evaluate turkey semen in vitro (for comprehensive review see [4]). With few exceptions, e.g., sperm motility [5,6, 7,8], there is limited evidence that the results of any of these procedures can be correlated with sperm fertility. DNA fingerprinting of offspring has been used extensively in many species and is a useful tool in determining paternity of progeny from females subjected to multiple consecutive matings [9,10,11]. Jones and Mench [12] were the first to utilize this technique to determine paternity of chicks from roosters of the same breed. The objective of this study was to determine the paternity efficiency of individual toms after heterospermic insemination through the use of DNA fingerprinting. Using standard semen quality tests, semen from toms subjected to DNA fingerprinting was evaluated to determine whether measurable differences between ejaculate quality or sperm characteristics were related to paternity efficiency. MATERIALS AND METHODS EXPERIMENTAL DESIGN Semen collection, pooling of ejaculates, dilution, and insemination were performed using standard industry methods [l]. Toms (n = 10/trial) were randomly placed in three groups. Semen was collected weekly for 3 wk, pooled by group, and used to inseminate 12 hendgroup. Ejaculates from individual toms were also evaluated for semen volume and sperm concentration; for viability using dual fluorescent stains SYBRPI and Calcein AMP1 [13]; and for membrane integrity using a hypo-osmotic ethidium bromide stress test [14]; they also received a subjective motility evaluation. Eggs were collected for 4 wk and candled at 7-10 days of incubation to determine fertility. Fertile eggs were incubated for a total of 28 days; blood was taken from toms, hens, and hatched poults and was frozen for DNA analysis. Paternity was determined by DNA fingerprinting. ANIMALS AND SEMEN COLLECTION Large White breeder toms and hen poults were purchased from a primary breeder and maintained under standard management conditions during brooding and growing periods. At 28 wk of age, toms were photostimulated by increasing light exposure from 12 hr Iight:12 hr dark to 14 hr light:lo hr daik to stimulate semen production. Semen was collected using the abdominal massage method [15] beginning at 30 wk of age. Toms that consistently produced at least 0.1 ml of thick white semedejaculate during the first three semen collections were selected for the study. Large White breeder turkey hens were photostimulated at 32 wk of age by increasing the duration of light exposure daily from 6 hr light:18 hr dark to 14 hr light:lo hr dark. Hens were inseminated once a week with pooled semen for 3 wk beginning 16 days post-photostimulation. SEMEN EVALUATION Semen was evaluated by various tests to determine whether a semen and/or sperm trait correlated with paternity could be identified. Semen from each tom was collected into a graduated conical tube and semen volume recorded. The concentration of sperm in each ejaculate was measured in a Klett Summerson colorimeter (Klett Mfg. Co., New York, NY [16]). The percentage of sperm moving in a rapid linear path was estimated subjectively by light microscopy. Using 10 pl of diluted semen on a slide at room temperature, progressive motility was scored on a scale of 0 to 5 with 0 sqpifying no forward movement and 5

5 216 PATERNITY EFFICIENCY representing rapid linear forward progression. To assess sperm viability, a combination of liveldead stains previously validated for use on turkey sperm was used [B]. SYBR-14, a membrane permeant DNA stain, and Calcein-AM (CAL), a membrane permeant nonspecific esterase substrate (FertiLiit Kit, Molecular Probes, Eugene, OR), stain only living sperm, producing bright green fluorescence of the nuclei (SYBR) or the entire cell (CAL) when excited at 488 nm. Propidium iodide (PI) stained membrane-damaged cells red. The SYBR-14 and CAL were dissolved in anhydrous dimethyl sulfoxide (Aldrich Chemical Co., Milwaukee, WI) at a concentration of 1 mg/ml. The PI was dissolved in Beltsville Poultry Semen Extender (BPSE, Continental Plastics, Delvan, WI) at 4 mg/ml. Aliquots of diluted semen (500 pl) were stained with 0.27pL of a SYBR-14 or CAL stock (original stock diluted 1:lO with DMSO for staining, 0.1 mg/ml) and 8 pl of PI stock. All samples were incubated at 37 C for 15 min. Immediately after incubation, samples were diluted 1:3 in phosphate buffered saline (PBS) and analyzed by flow cytometry. For both SYBR/PI and CALPI viability assessment, 10,OOO individual sperm were assessed by flow cytometry for each replicate. To determine membrane integrity, a hypoosmotic ethidium bromide stress test was used [14]. Semen (1OpL) was added to a 2 ml solution of ethidium bromide (12.5pg/mL) in PBS (296 mosm/kg) or hypotonic PBS (60 mosdkg) and mixed gently. The mixed sample was placed in a fluorometer (Fluoro-Tec, American Research Products, Beltsville, MD) for 2 min to get an initial reading. To disrupt all sperm membranes, u)pl of digitonin (5pg/mL ethanol) was then added and mixed, and the same sample was placed in the fluorometer and a reading taken 2 min later. The initial and final readings multiplied by 100 gave the percentage of nonviable sperm. The difference between the iso-osmotic and the hypo-osmotic viability was the percentage of sperm that had intact membranes. FLOW CYTOMETRY Quantitative assessment of fluorescently stained sperm was performed using an EPICS Profile I1 (Coulter Electronics, Inc., Hialeah, FL). The flow cytometer utilizes an air-cooled Argon laser (488 nm) and is equipped with the PowerPak option to provide for three-color fluorescence detection in addition to the side and forward light scatter parameters. Data were collected as log of fluorescence on 10,OOO sperm per sample. The green wavelength fluorescence (LFL1) was collected through a 525 nm band pass filter, while the red fluorescence parameters, fluorescence 2 (LFL2) and fluorescence 3 (LFL3), were gathered through 575- and 635-nm band pass filters, respectively. Data were analyzed using the Coulter Histogram Analysis program. GENOMIC DNA ANALYSIS AND PATERNITY EVALUATION Blood was collected by standard brachial venipuncture in 0.5 M EDTA, aliquoted, and frozen at -20 C. Genomic DNA was isolated from each 50 pl aliquot as described elsewhere [17]. Eight micrograms of genomic DNA from each candidate sire and progeny was digested by Hinf I according to standard protocol [18]. Prior to digestion, DNA mixes from the female parents from each trial were made by combining 1 pg of DNA from each of twelve individuals. Digested DNA samples from individual samples and pools were electrophoresed in 0.8% agarose gel for 36 hr and blotted onto nylon membranes after standard depurination and denaturation [19]. The blots were hybridized using (GATA)5 as the probe after end-labeling as previously described [19]. After hybridization, membranes were washed at low stringency and then exposed to film for 6-12 hr. To determine paternity, only males with progeny-specific bands not present in the hen pool were considered candidate sires. Among candidate sires, paternity was inferred if the tom had a bandsharing frequency with a poult equal to or greater than 50% [u)]. Toms without any of the poultspecific bands were excluded as a potential sire of a given poult. STATISTICAL, ANALYSIS Semen evaluation data was analyzed by ANOVA using the least squares procedure and the General Linear Models and Correlation Procedures of SAS [21]. Data expressed as percentages were arc sin transformed before analysis.

6 DONOGHUE et al. Research Report 217 RESULTS AND DISCUSSION Following heterospermic inseminations with pooled ejaculates from multiple toms, the majority of the progeny were sired by few males. For Trials 1-3, seven, nine, and ten males, respectively, produced semen and were included in the study. In Trials 1 and 2, paternity efficiency was highly skewed, with only one or two males producing a majority of the offspring. In llial 1, one tom produced 37 of the 70 poults tested (52.9%); in Trial 2, two toms produced 83% of poults analyzed (Figure 1). In Trial 3, three toms produced 62% of the progeny. In Trials 1 and 3 all toms produced progeny; however, in Trial 2, three of nine toms produced no poults. Previous assumptions were that the high quality sperm from different males in pooled semen were equally capable of producing offspring using standard industry practices. The present study, however, demonstrated that relatively few toms sired a high percentage of the progeny. Of the 26 toms contributing to the pooled ejaculates, six (23%) sired more than 60% of the 145 poults. Conversely, 14 toms (54%) sired 0 to 3 poults. Heterospermic inseminations have been used in poultry to elucidate the mechanisms of oviductal sperm storage [ll, 221. In these studies, major phenotypic traits such as dwarfism, plumage color, or comb shape established the paternity. Heterospermic insemination has also been used extensively in roosters to determine paternity [23,24,25,26]. However, these studies evaluated sperm competition between few males, unlike the present study, in which multiple ejaculates were pooled. One of the goals in the current study was to determine whether an ejaculate trait or sperm characteristic would correlate with paternity. None of the semen characteristics evaluated were consistently related to paternity. No correlation was observed between semen volume and paternity efficiency (Tables 1, 2, and 3). For toms that averaged 0.5 ml of semen per ejaculate, only one tom produced a high proportion of offspring; the other five toms produced less than 6% of the progeny in their respective trials.?tvo toms producing the lowest ejaculate volumes produced 42% of the offspring. Ejaculate color and semen volume are traits routinely used in the industry to cull toms from a breeder flock [27l. White semen is used because yellow semen has been associated with reduced fertility (see review [24]). Semen volume can vary due to the collector but generally ranges from 0.1 to 0.6 ml for each ejaculate. Since viscous white ejaculates are assumed equally fecund, toms producing low semen volumes (< 0.2 ml/ejaculate) are culled on commercial farms. In our study ejaculate volumes varied between toms and when pooled for inseminations did not influence paternity (Tables 1, 2, and 3). For example, average ejaculate volume from one tom was low (0.17 ml), yet this tom produced 37% of the progeny in Trial 2. In all three trials, only five toms averaged more than 0.5 ml of semen, yet four of these toms sired less than 6% of the progeny. Such observations clearly show that semen volume is not a good indicator of potential fecundity. In addition to visual examination and semen volume determination, sperm concentration is frequently estimated on commercial farms. With the development of stud farms, which are tom-only farms dedicated to optimizii semen production and distribution to hen farms, the insemination of hens based on semen dose instead of semen volume has been adopted by alarge portion of the industry [25]. Yet sperm concentration is measured in a pooled ejaculate, not for individual toms. In our study, after pooling several ejaculates without correcting for concentration between toms (as practiced on modern stud farms) we observed no correlation between paternity and ejaculate concentration of individuals even though sperm concentration ranged from 3.8 to 7.4 billion sperm/ml (Tables 1,2, and 3). In other studies, sperm numbers have been shown to influence paternity after heterospermic insemination [24,26,27,28]. Because the concentration of turkey sperm is extremely high (ejaculate concentrations in the billions/ml) compared to the ejaculates of other species, differences in total sperm numbers between toms might not have varied enough to influence sperm competition. That is, the number of sperm from each tom may have far exceeded the minimum number of sperm necessary for fertility. Use of a substantially decreased sperm concentration (millions) may have yielded the correlations between paternity and number of sperm inseminated observed in other studies.

7 218 PATERNITY EFFICIENCY Trial 1 45 m 40.- C L 35 P * 0 20 L 0 15 D % Toms Trial 2 ul % 14 C L 12 a g! 10 0 r 8 0 t 6 Q f 4 = 2 0 Toms Trial h IO v) e 0 8 r O 6 L Q a 4 E 22% 2.2% 2.2% $ 2 0 Toms FIGURE 1. Paternity of offspring from pooled turkey semen

8 DONOGHUE et al. Research Report 219 TABLE 1. Eiaculate characteristics and semen quality assessment of individual toms in Trial 1 ranked by k1.0 35k k k kl.O k kO k k k k k k k k k k k k k k k k k k k k k k k k k k k k8.7 SDerm viabilitv and sderm Dlasma mem- I brane integrity are semen quality characteristics often examined. Utilizing dual-stained sperm and flow cytometry, thousands of sperm can be objectively assessed in a relatively short time period. In this study, even though the proportion of live cells in ejaculates from individual toms ranged from 50 to 80%, no relationship between sperm viability and paternity was observed (Tables 1,2, and 3). Similarly, no relationship between membrane integrity and paternity was observed (Tables 1, 2, and 3). Previous fertility trials that adjusted the number of viable sperm based on hypo-osmotic stress test results also did not influence hen fertility [14]. The tom producing the most progeny in Trial 1 also had the highest sperm motility estimates, and paternity efficiency and sperm motility were positively correlated in this trial (P c.05, Table 1). However, this was not the case for Trials 2 and 3, in which the males with the highest sperm motility produced few or no offspring (Tables 2 and 3). Several investigators have reported correlations between subjective motility estimates and fertility [29, 30, 311. Comparing roosters of different breeds in a heterospermic insemination experiment, Allen and Champion [a] found a

9 220 JAPR PATERNITY EFFICIENCY TABLE 3. Ejaculate characteristics and semen quality assessment of individual toms in Trial 3 ranked by *SYBR-14, a membrane permeant DNA stain and Calcein-Ah4 (CAL), a membrane meant nons cifc esterase substrate stain on1 livin sperm, prpducin baght green fluorescence of the nuclei SGR) or the en% cell (CAL) when excked at 4dnm. {ropidiurn iodide &'I) stained membrane-damaged cells rei. positive correlation between motility (as subjectively scored on a microscope slide) and fertility (R =.72). Assessment of sperm motility of individual toms is not standard practice on breeding farms. This may occur because of the subjectiveness of motility estimates and the inability to reliably compare defined motility values between investigations, or because motility appeared convincing in one trial but was not repeatable in subsequent trials, as occurred in the present study. Although the means for identifymg high and low fecundity in toms are currently available, it would be impractical to use DNA fingerprinting for commercial identifcation of paternity efficiency due to: 1) the expense and technical expertise required for the procedure; 2) the time delay between insemination-hatch and paternity analysis of poults (a minimum of 5 wk), which would result in a substantial loss of a sire's reproductive lifetime; and 3) the need to collect blood samples from all potential sires, hens, and poults for DNA analyses. The identification of a semen trait which could be correlated with hgh or low rates of paternity would have important management and financial benefits. The selection of young poults based on semen traits which correlate with future fecundity may allow a reduction in the number of toms raised and placed on the stud farm and even an increase in the interval between successive inseminatons. A philosophy exists in turkey management that semen is pooled from many toms in order that males of high fertility potential will compensate for toms with low fertility potential. However, the effect of semen pooling on paternity efficiency had not been demonstrated scientifically until now. Toms with substandard fertility are managed throughout their breeding lifetime, yet they could be eliminated if identified early in production. A method to systematically cull toms of low or no paternity potential could dramatically alter commercial tom management. CONCLUSIONS AND APPLICATIONS 1. Paternity efficiency was highly skewed, with few toms producing a majority of progeny. 2. Semen parameters evaluated, some of which are used routinely by the turkey industry, were not good predictors of paternity. 3. Although some individuals are not contributing to progeny, the current commercial practice of pooling ejaculates from toms does not take this into consideration.

10 Research Report DONOGHUE et al Bahr, 1. M. and M. R Bakst, Chapter 18 Poultry. Pages in: Reproduction in Farm Animals. 5th Edition. E.S.E. Hafez, ed. Lea & Febiger, Philadephia, PA. 2. Dziuk, PJ., A review Factors that influence the proportion of offspring sired b a male following heterospermic insemination. Anim. Leprod. Sci Baht, M.R. and H.C. Cecil, Evaluation of turke semen. Pages in: Proc. 2nd Intl. Symp. Turkey %prod, Raleigh, NC. 4. Bakst, M.R and H.C. Cecil, Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. Poultry Sci. Assn., Savoy, IL. 5. Wisharf, GJ., Techniques for semen quality determination. Pages in: Proc. 3rd Intl. Symp. on Turkey Reprod., Raleigh, NC. 6. Froman, D.P. and DJ. McLean, Objective measurement of sperm motility based upon sperm penetration of Accudenz. Poultry Si. 75:? Froman, D.P., AJ. Feltmano, and DJ. McLean, Increased fecundity resulting from semen donor selection based upon sperm motility. Poultry Sci Donoghue, AM., D.R Holsberger, D.P. Evenson, andd.p. Froman, Semen donor selection by h a sperm mobility increases fertili and sperm storage in the turkey hen. J. Andrology 19:29? Burke, T., N.B. Davies, M.W. Bruford, and BJ. Hatchwell, Parental care and mating behavior of polyandrous dunnock (Prunella modulans ) related to paternity by DNA fingerprints. Nature Gibbs, H.L, PJ. Weathershead, P.T. Boa& B.N. White, LM. Tab& and DJ. Hoysak, Realized reproductive success of polygynous red-winged blackbirds revealed by DNA markers. Science Birkhead, T.R, GJ. Wishart, and J.D. B%pens, Sperm precedence in the domestic fowl. Proc. Royal SOC. London Jones, M.EJ. and J.A Mench, Behavioral correlates of male mating success in a multisire flock as determined by DNA fingerprinting. Poultry Sci Donoghue, A.M., D.L Gamer, DJ. Donoghue, and LA Johnson, Viability assessment of turkey s erm usin fluorescent staining and flow cytometry. Boultry~ci.54:1191-1~~). 14. Bakst, M.R, H.C. Cecil, and TJ. Sexton, Modification of the ethidium bromide exclusion roce dure for evaluation of turkey semen. Poultry Sci. 79:& Burrows, W.H. and J.P. Quinn, The collection of s rmatozoa from the domestic fowl and turkey. Poultry E 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: REFERENCES AND NOTES 17. Bartlett, J.R, C.P. Jones, and EJ. Smith, Linkage analysis among endogenousviral element 1, blue e shell, and pea comb loci in chickens. J. Heredity 8p Sambrook, J., EF. Fritsch, and T. Maniatis, Molecular Cloning: A Laborato Manual. 2nd Edition. Cold Spring Harbor Laboratory Yress, New York, NY. 19. Smith, E.J., H-B. Huang, M. Hsei, and R. Zahorchak, Two turkey genomic fragments containing minisatellite repeats reveal lymorphic DNA fmgerprints. him. Genetics 28:245-g Wetton. J.H.. R.E. Carter. D.T. Parkin. and D.Walters, 1~71 Demographic study of a wild house sparrow population by DNA fingerprinting. Nature SAS Institute, SAS User s Guide. Version 5 Edition. SAS Institute, Inc., Cary, NC. 22. Compton, M.M., H.P. Van Krey, and P.B. Siege& The filling and emptying of the uterovaginal spermhost glands in the domestic hen. Poultry Sci. 57: Etches, RJ., RB. Buckland, and RO. Hawes, The effect of the genes for rose comb and polydacon sperm transport in the hen s oviduct. Poultry Sci s 24. MarUn, PA, TJ. Reimcrs, J.R Lodge, and PJ. Dduk, The effect of ratios and numbers of s rmatozoa mixed from two males on proportions of ofgring. J. Reprod. Fertility Martin, PA. and PJ. Dziuk, Assessment of relative fertility of males (cockerels and boars) by competitive mating. J. Reprod. Fertility Klrby, J.D.,D.P.Proman,H.N.Engel, Jr.,andP.E Bernier, Decreased sperm survivability in subfertile Delaware roosters as indicated b comparative and competitive fertilization. J. Reprod. 8ertility 86: Christensen, V.L, Semen collection and dilution. Pages 1-5 in: Techni ues for Semen Evaluation, Semen Storage, and Fertility%etermination. M.R Bakst and H.C. Ceal, eds. Poultry Science Assn. Inc., Savoy, IL. 28. Men, CJ. and LR Champion, Competitive fertilization in the fowl. Poultry Sci. 34: Whn, H.R, N.P. Piesco, ER Miller, and W.G. Nesbeth, Prediction of the fertility potential of broiler breeder males. World s Poultry Sci. 35: Wishart, GJ. and F.H. Ross, Characterization ofa spectrophotometric technique for theestimation of fowl and turkey motility. Gamete Res. 11: Holsberger, D.R, AM. Donoghue, D.P. Froman, and MA. Ottingcr, Assessment of ejaculate quality and sperm characteristics in turkeys: Sperm mobility phenotype is independent of time. Poultry Sci. T7: ACKNOWLEDGMENTS Thanks to Denise Holsberger and Valorie Akuffo for technical assistance and to Wayne Smoot, Dale Bushling, and Doug Greenfield for semen collection, artificial insemination, and care of animals.