Assessing Potential Fertility of Individual Males: Evaluation of Sperm Characteristics and Function in Turkeys

Transcription

1 Assessing Potential Fertility of Individual Males: Evaluation of Sperm Characteristics and Function in Turkeys Ann M. Donoghue, Germplasm anit Gamete Physiology Lab., ARS, USDA Beltsville, MD Introduction Reproductive management in turkeys is extremely labor-intensive since artificial insemination (AI) is used exclusively for reproduction. Furthermore, if broiler breeders continue to increase in body weight and skeletal frame, AI may be the only economical means to produce hatching eggs. In livestock production systems where AI is practiced, extensive semen analysis is fundamental to sire selection and reproductive management. However, for production of the commercial turkey, evaluation of semen from individual toms is limited to visualization of semen color, measurement of ejaculate volume and possibly sperm concentration. Sire selection is not a major consideration for commercial operations. Management practices dictate pooling of semen from toms to provide adequate semen volume for the large numbers of hens requiring AI on a weekly basis. It is generally assumed that sperm in good quality semen from all toms are equally fecund. However, paternity efficiency after pooling multiple ejaculates has not been previously determined in the turkey. This paper will outline why it may be important to evaluate males as individuals and how we can use advances made in the understanding and measurement of sperm function to improve reproductive efficiency in turkeys. Evaluating Paternity Efficiency While it is known that sperm competition exists, the degree to which one male's sperm fertilizes an ovum relative to another male remains unknown. We determined individual male fecundity and attempted to establish a relationship between male fecundity and semen characteristics (Donoghue et al., 1998a). Semen was collected weekly for 3 weeks (n= 10 toms/trial), pooled by group and used to inseminate 12 hens/group. Ejaculates from individual toms were also evaluated for semen volume, sperm concentration, viability (using dual fluorescent stains), membrane integrity (using ethidium bromide hypo-osmotic stress test), and a subjective motility evaluation. Eggs were collected weekly for 4 weeks and candled at 7-10 days of incubation to determine if they were fertile. Fertile eggs were incubated for 28 days, hatched poults were bled, and blood samples frozen until DNA analysis was performed. Blood samples were also collected from toms and 59

2 hens and frozen until analysis Paternity efficiency of toms was determined by DNA fingerprinting in collaboration with Dr. Ed Smith at Tuskegee University. In two trials, paternity efficiency was highly skewed with only one or two toms producing a majority of the. offspring (.Table 1). In Trial 1, one tom produced 37 of the 70 poults tested (52.9 % of progeny analyzed) and in Trial 2, two toms produced 83.4 % of poults evaluated. In the third trial, all toms produced progeny yet four of ten toms produced 75.5 % of the offspring. The semen parameters evaluated in this study, some of which are used routinely by the turkey industry, were poor predictors of paternity. We can conclude that paternity efficiency following heterospermic insemination is an issue that should be addressed Although the means for identifying high and low fecundity in toms is available, it would be impractical to use DNA fingerprinting for commercial sire selection due to: 1) the expense and technical expertise of the procedure; 2) the time delay between insemination-hatch and paternity analysis of poults (a minimum of 5 weeks) resulting in a substantial loss of a sires' reproductive lifetime; and 3) the need to collect blood samples from all potential sires, hens, and poults for DNA analysis. Since artificial insemination relies on the collection of semen from all toms in a breeder flock, a readily detectable sperm trait strongly associated with fecundity could be easily incorporated into breeder tom management. However, using standard semen quafity tests, evaluation of semen from toms subjected to DNA fingerprinting resulted in no detectable difference in ejaculate quality or sperm characteristics between toms producing numerous offspring and those producing few to none (Donoghue et al., 1998a). Over the last several years a concentrated effort has been made to develop and evaluate tests which quantitate sperm function in poultry (Wishart, 1993, 1995; Bramwell et al., 1995; Froman and McLean, 1996). Since many factors influence sperm function, reviewing the steps sperm must successfully negotiate through the hens tract may be key to understanding sire potential. Review of Sperm Function Sperm must be motile and survive the environment of the vagina to reach the sperm storage tubules (SST), crypts which store sperm in the hen for extended periods of time. This "reservoir" of sperm within the SST insures that sperm are available between inseminations and are the key to sustained fertilization. Upon release from the SST and transport to the infundibulum, the site of fertilization, sperm must be capable of binding to and penetrating the perivitelline layer (a single acellular investment enveloping the ovum at ovulation) and then fertilize the ovum. Success at every step is imperative for fertilization. 6O

3 Table 1. Paternity Efficiency After Pooling Ejaculates from Multiple Toms Determined by DNA Fingerprinting. Tom Progeny Percent Paternity (%) Trial 1-total of 70 poults Trial 2-total of 30 poults Trial 3-total of 45 poults

4 What sperm traits can be evaluated? Although simplifying the process, there are some key traits which can be estimated. First, sperm must be motile to traverse the vagina and reach the SST. Sperm motility can be measured outside of the hen giving us an indication of motility within the hen. Second, since sperm storage in the SST is critical to.tong term fertility, this can also be quantitatively evaluated. By isolating the perivitelline layer of freshly laid eggs and observing the holes created by sperm hydrolyzing a path through this layer we can estimate: 1) the number of sperm present at the site of fertilization and 2) the duration of fertility. The sperm hole number is correlated to number of sperm stored in the SST allowing an indirect measure of SST capacity. Finally, sperm have to bind and penetrate the egg. This can be evaluating using a solubilized extract of perivitelline layer to assess sperm binding in vitro. Sperm Motility Assessment Sperm are vehicles which carry DNA to the ovum and as such the "motor" is important. Sperm motility is a good indicator of sire potential and has been used by several investigators to predict potential fertility. Historically, the swirling movement of sperm placed on a microscope slide has been used as a subjective estimate of sperm motility. The Swirl Method has shown good correlation with fertility in chickens and turkeys (Wilson et al., 1979). An important distinction of some of the newer methods of motility assessment is the objectivity of the tests, that is, each semen sample is scored by an instrument instead of a technician, reducing the subjectivity of semen assessment. One of the first objective sperm motility tests available for poultry sperm was the Spectrophotometer Technique developed by Wishart and Ross (1985). A very promising approach for objective sperm motility analysis recently has been developed for rooster sperm (Froman and McLean, 1996) and modified for turkeys (Donoghue et al., 1998b). The Sperm Mobility Test is based upon the ability of sperm to swim into a dense, inert non-toxic diluent called Accudenz. This assay is performed at body temperature (41"C), and requires sperm to be mobile to penetrate into a solution, possibly mimicking some of the environment sperm are exposed to in the hen's reproductive tract. Male-to-male variation in sperm mobility phenotype has been estimated using the Sperm Mobility Test and shown repeatedly to be a normally distributed trait (Froman and McLean, 1996; Froman et al., 1997; Froman and Feltmann, 1998; Holsberger et al., 1998). Froman contends that sperm mobility is a quantitative trait and most importantly, sperm mobility is a determinant of fecundity (Froman and Feltmann, 1998). When toms were selected out of a flock based on the extreme limits of sperm mobility, 62

5 fertility was higher or lower respective of sperm phenotype (Table 2). Table 2. Effect of Sire Selection on Fertility after Pooling of Ejaculates by Sperm Mobility Phenotype and Weekly Inseminations in Two Trials Treatment Eggs Fertility AI Dose High Mobile a t 150 million Low Mobile _ _ million High Mobile b 3, t 75 million Low Mobile b 3, million From Donoghueet al, 1998b"Tenweektrial, bsixteenweek trial. tdiffers from Low Mobilewithin trial, P < When sperm mobility was measured in roosters over 20 weeks (Froman et al., 1997) and in toms over 22 weeks (Holsberger et al., 1998) it remained consistent within phenotype. This is an important finding, since sire-selection should take place early in production and the benefit of improving semen quality maintained over the entire breeding period. When evaluating individual toms over the course of semen production, Holsberger and co-workers (1998) found no relationship between sperm mobility phenotype and several semen evaluation parameters including semen volume, concentration, sperm viability and sperm membrane integrity. Whereas the sperm mobility test was predictable of fertility, none of the other semen/sperm characteristics correlated with this trait. The Sperm Mobility Test has the potential for use as a method of predicting the fertilizing ability of potential sires where other semen evaluation tests have failed. Using the Sperm Mobility Test in conjunction with fertility assessment of broiler breeder males (5 lines/ roosters), John Kirby's group at the University of Arkansas found that sperm mobility was highly predictive of observed fertility (R= ; Rhoads et al., 1998). In addition, Froman and co-workers (1997) have demonstrated that increasing the AI dose of average mobility roosters does not improve fertility as compared to hens inseminated with semen from high mobility males. An advantage of the Sperm Mobility Test is that it is simple, requires little technical expertise and is consistent over the reproductive life of a tom. Its potential for on farm use is good, although it needs to be modified for farm application, a project that is currently in progress in collaboration with Nicholas Turkey Breeding Farms and Hybrid Turkeys Inc. 63

6 Quantitation of Sperm Storage in the Hen Sperm storage in the hen is a critical component to sperm function since the stored sperm maintain fertility between inseminations. Over the last ten years an intensive effort has been made to quantitate sperm in the hens' reproductive tract in order to estimate flock and individual hen fertility. Methods have been developed which use the freshly laid egg to gain insight into sperm numbers in the SST (see review, Wishart, 1995; Brillard and Bakst, 1990). One such assay relies on determining the holes created by sperm hydrolyzing through the perivitelline layer of ova in the infundibulum (approximately 24 to 26 hours before oviposition; Bramwell et al., 1995). The sperm hole number can be correlated to the number of sperm stored in the SST, allowing an indirect measure of SST capacity (Wishart, 1995). Although this assay has been used extensively to evaluate and predict flock fertility, it also has potential to indicate differences in toms. When semen from toms of high, average and low mobility (based on Sperm Mobility Test results) was pooled by group and used to inseminate hens, the mean number of holes observed for good and average mobility toms were almost four times higher than that of poor toms, indicating that sperm storage in the hen and the number of sperm at the site of fertilization is reduced in toms with low mobile sperm (Donoghue, Holsberger and King, preliminary data, Table 3). Table 3. Sperm Holes Observed in Eggs from Hens Inseminated with Sperm from High, Average or Low Mobility Phenotype Toms Number Number of Phenotype of Eggs Sperm Holes Good a Average a Bad I- 4.1 b a'bcolumnswithdifferentsuperscriptsdiffer,p<.05. Includes1 weekof eggs, n=8 hens/trt. The sperm hole assay is a great tool for assessment of sperm function in the hen. When used as a measure of sire selection, one must keep in mind the influence of the hen in the results. This technique is quick to master but is time consuming. It may be used more to confirm the validity of other sire selection assays (a shown with the Sperm Mobility Test) or as a quality control measure of flock fertility after sire selection. 64

7 Sperm Binding Assay The ability of sperm to bind to the perivitelline layer is a pivotal step in fertilization. A sperm-binding assay has been developed in order to measure this ability. This assay utilizes microwell plates coated with an extract of chicken perivitelline layer. Sperm can bind to the microwell and can be quantified, allowing for detection of differences in individual roosters or toms. Differences in sperm binding to the plate correlates with fertility (Barbato et al., 1998). When F._,.,.,_.,_...A,.,I...,...,,._,.--,.,_._..- used to screen toms, the Chulllfled u Geod or Bid _ on Sperm Binding _ulay percentage of sperm binding " r. ---_--_.,".-L--A. i--/ : -r---_--..:. _ I,,,qj. individuals. When toms were " to the plates differed among T -],,_.,._.. _ sb _, selected based on percent _,, "'1." " sperm binding and used to _.,, inseminate hens on a weekly AI schedule (75 million ** --*'*' sperm/dose) ' differences in O I I"" "" l l I I l l I I I I I fertility were apparent by the.,,_.,' ' " ' ' ',,..,.' ' '* " " " " 8th week of AI (Figure 1). Fertility from hens inseminated with sperm from poor-binding toms continued to decline through the end of the study whereas the fertility of the good-binding sperm group remained high. It is well documented that fertility declines over the course of the production period. If data had been collected only during the first portion of this study, the effects of the poor sperm binding group would have been missed. This is an important point when analyzing sperm assessment tests, evaluating fertility over an extended period of time could result in very different conclusions from that of limited fertility trials. The advantage of the sperm-binding assay is that unlike the sperm hole assay, the sperm binding assay eliminates the hen effect, while enabling the identification of toms with low fertility. The binding assay requires some technical skill and time, and utilizes a microplate reader. It is currently being adapted and simplified for use in commercial flocks (Barbato et al., 1998). Conclusions The impact of sire selection based on a test which correlates fertilizing potential would be extremely beneficial to the turkey industry. with sperm The ability 65

8 to differentiate toms with high fertility potential would provide a rational, objective reason to cull toms with low fertility from a flock. Technologies, such as DNA fingerprinting provide valuable information as to how sperm function in a pooled sample and this information could alter the way toms are currently managed. If males with substandard fertility do not contribute to the production of offspring they could be evaluated early in production and removed from the breeding flock. If we can determine what makes a tom more fecund during the sperm selection process in the hen, these traits could be translated into methods of screening sires. The Sperm Mobility Test, the Sperm-Hole Assay and the Sperm-Binding Assay have been shown to predict fertility and show promise as management tools for sire selection. As we learn more about the mechanisms of sperm function we can use this knowledge to select individual sires and improve the overall efficiency of managing flocks. Acknowledgments The author thanks Denise Holsberger and Laura King for their efforts in the research contained in this manuscript, collaborators Ed Smith, Tuskegee University and Murray Bakst, GGPL, ARS, Beltsville for the paternity study; David Froman, Oregon State University, and John Kirby, University of Arkansas, for data and for collaboration with studies using the Sperm Mobility Test; and S.P. Gill, Rupert Amann and Roy Hammerstedt, Biopore, Inc. for the sperm binding work. The author thanks Paul Marini, David Harry and staff of Nicholas Turkey Breeding Farms and Joe Darden and staff of Hybrid Turkeys for collaboration in field trials. Thanks to Murray Bakst and Laura King for critical review of the manuscript. This work is partially funded by U.S. Poultry and Egg Association, grant #340. References Barbato, G. F., Cramer, P.G and Hammerstedt, R.H A practical in vitro sperm-egg binding assay which detects subfertile males. Biol. Reprod. 58: Bramwell, R.K., Mark, H.L. and Howarth, B., Quantitative determination of spermatozoa penetration of the perivitelline layer of the hen's ovum as assessed on oviposited eggs. Poult. Sci. 74: Brillard, J.P. and Bakst, M.R., Quantification of spermatozoa in the spermstorage tubules of turkey hens and the relation to sperm numbers in the perivitelline layer of eggs. Biol. Reprod., 43:

9 Donoghue, A.M., M.R. Bakst, P. Drummond, S. Haqque and E. J. Smith. 1998a. Paternity efficiency in turkeys differs extensively after heterospermic insemination. Proc. Inter. Symp. Spermatology (in press). Donoghue, A.M., Holsberger,D.R., Evenson, D.P. and Froman, D.P. 1998b. Semen donor selection by sperm motility assessment influences hen sperm storage and fertility in turkeys. Andrology 19: Donoghue, A.M., Gill, S.P. and Amann, R.P Influencing fertility in turkeys:tom selection by sperm-binding assessment and improved binding with synthetic protein in fresh and stored semen. Poult. Sci. 76:23. Froman, D.P. and McLean, D.J Objective measurement of sperm motility based upon sperm penetration of Accudenz. Poultr. Sci. 75: Froman, D.P., Feltmann, A.J. and McLean, D.J Increased fecundity resulting from semen donor selection based upon in vitro sperm motility. Poult. Sci. 76: Froman, D.P. and Feltmann, A.J Sperm mobility: A quantitative trait of the domestic fowl (Gallus domesticus). Biol. Reprod. 58: Roads, M.L., Washington, J., Froman, D.P. and Kirby, J.D Use of the sperm mobility assay to predict sperm fertilizing ability in broiler males. Poult. Sci. in press. Wilson, H.R., Piesco, N.P., Miller, E.R. and Nesbeth, W.G Prediction of the fertility potential of broiler breeder males. Wold Poult. Sci. 35: Wishart, G.J. and Palmer, F.H Characterization of a spectrophotometric technique for the estimation of fowl and turkey motility. Gamete Res. 11: Wishart, G.J. and Ross, F.H Correlation of the fertilising ability of semen from individual male fowls with sperm motility and ATP content. British Poult. Sci. 27: Wishart, G.J., Techniques for semen quality determination. Proc. Third Inter. Symp. on Turkey Reprod. pp Wishart, G.J., New approaches to evaluating male and female fertility. In: M.R. Bakst and G.J. Wishart (Editors), First International Symposium on the Artificial Insemination of Poultry, Poultry Science Assoc., pp

10 Question #1: Dr. Surinder Gill Can we pick good toms with the mobility assay and further screen them with the binding assay? Will these toms have better fertility than those picked by either of these two methods? Answer: Ann Donoghue Yes. Although results with both assays are fairly consistent in identifying the same poor males, we have observed differences in males that ranked as either high binders or high mobile. Ranking toms by the mobility assay and following up with the binding assay, as you suggest, might identify the elite males of a flock with regard to measurable sperm function. Question #2: Dr. Steve Lerner There is good evidence that from a random group of toms, only a couple produce the majority of offspring. If you pool only "good" toms, would the distribution of offspring change? Answer: Ann Donoghue At this point I would be speculating but I believe regardless of the contents of a mixed ejaculate sperm competition will occur. This is based on several studies in many species and nicely reviewed by Phil Dziuk (Anim. Reprod. Sci :65-88). However, what I suggest is that possibly by pooling only good toms the paternity efficiency would be closer to equal between toms than in a random pool. Question #3: Dr. Charles Stuber What is the heritability of fertility (sperm characteristic) traits? Answer: Ann Donoghue This is a good question and one that needs to be explored. Dave Froman has received a NRI grant and is currently evaluating the heritability of sperm mobility in roosters so hopefully this information will be available soon. Guy Barbato and Roy Hammerstedt have addressed this issue with the sperm binding assay in their recent Biology of Reproduction paper and Guy will discuss this in detail at the up coming symposium on Managing Poultry Reproduction at the Poultry Science meeting this summer. 68

11 Question #4: Dr. Bob McKay How repeatable is the fingerprinting data identifying sires contributing the most progeny? If you take a different set of females with the same sample of males, would they rank the same? Answer: Ann Donoghue Obviously there will be a hen effect. In the study I presented we did have multiple hens and the same few toms produced a majority of the offspring. Therefore, I believe we would see the same effect on another set of hens. This is an area that we believe is important so we do plan on continuing this work and repeating our study. 69