PHYSIOLOGY AND REPRODUCTION. Fertility Rate of Daily Collected and Cryopreserved Fowl Semen

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1 PHYSIOLOGY AND REPRODUCTION Fertility Rate of Daily Collected and Cryopreserved Fowl Semen A. VAN VOORST and F. R. LEENSTRA Institute for Animal Science and Health (ID-DLO) "Het Spelderholt", P.O. Box, AA Beekbergen, The Netherlands ABSTRACT Semen was collected for consecutive d individually from experimental broiler breeder males that had not been massaged for d. The semen was mixed and diluted with the Beltsville Poultry Semen Extender with dimethylsulfoxide (DMSO) as cryoprotectant and cryopreserved. After thawing of the semen, hens were inseminated and fertility over wk after a single insemination with the cryopreserved semen was determined. The overall fertility rate in this experiment (8 to 9%) was high, compared with the generally reported fertility rate of frozen-thawed fowl semen. The fertility rate of the semen collected on Days and was significantly higher than that of semen collected on Day or, indicating that frequent collection over the -d period enhances the fertility rate of the semen. (Key words: fowl, semen, cryopreservation, fertility rate) INTRODUCTION Cryopreservation of semen is an effective method for genetic conservation in farm animals, provided the frozen-thawed semen has sufficient fertilizing capacity. In domestic fowl several procedures for freezing and thawing of semen have been developed. The diluent, the cryoprotectant, freezing and thawing rate, absence of material deleterious to fertility, and fertilization capacity after thawing determine successful cryopreservation of sperm (Buss, 99). Cryopreservation of domestic fowl semen results in a decreased fertility rate when compared with fresh, unfrozen semen (Lake, 98; Bellagamba et al., 99). Hammerstedt and Graham (99) summarized recent research on fertility of frozen-thawed fowl semen. They found a fertility of % using dimethylsulfoxide (DMSO) and % using glycerol. The freezing and thawing protocol using DMSO developed by Sexton (98) allows for insemination directly after thawing Received for publication April, 99. Accepted for publication September, Poultry Science :- from straws used for storage (Haije, 99). This improves field use compared with methods using glycerol as cryoprotectant, which has to be removed before insemination. Semen quality is a determining factor in effective cryopreservation. Froman (99) observed in a unique line that spermatozoa degenerate during their stay in the deferent ducts. These degenerations were apparent on weekly collection of semen and disappeared on daily collection of semen over a period of d. Fewer degenerated spermatozoa are found in semen of males ejaculated relatively frequently (de Reviers and Williams, 98). In male passerine birds, Quay (98) found a spontaneous continuous release of spermatozoa from the excurrent ducts. This might point at removal of aged spermatozoa. Shorter stay of matured spermatozoa in the different ducts possibly has a positive effect on fertilizing ability. This might be obtained by daily ejaculation. Haije (99) found that daily collection of semen over a period of d did not reduce volume and concentration of the ejaculate nor the quality of the spermatozoa. Downloaded from at Wageningen UR Library on September 9,

2 FERTILITY OF FROZEN FOWL SEMEN Visible and invisible degeneration of spermatozoa, due to aging of the spermcells in the deferent ducts, might enhance the negative effects of cryopreservation on fertilizing capacity. In this experiment, therefore, the possible effects of aging of spermatozoa were examined by studying fertility rate of daily collected and cryopreserved semen. Better fertility results after freezing and thawing will indicate improved quality of the ejaculated semen. MATERIALS AND METHODS The experiment consisted of four replicates, carried out during consecutive wk. Ten males were used in Replicates and and different males in Replicates and. In each replicate trial, semen was collected for consecutive d from the same males. Chickens Twenty broiler breeder males of a Spelderholt selection line (FC line, Leenstra, 988) were used. The males were 9 wk of age at the start of the experiment. Seventytwo White Leghorn females were inseminated to determine fertility rate. They were wk of age at the start of the experiment. All birds were housed in individual cages. Daily lighting was h light and h darkness. All the birds received a standard layer diet (.% crude protein and,8 kcal ME/kg). The females had feed and water available for ad libitum consumption, the males were restricted to a feed intake of g/d and had water available during h/d. Collection and Dilution of Semen Semen was collected according to the massage technique of Burrows and Quinn (9). At the start of each replicate trial the males had not been massaged for d. The semen was collected after a single massage. The ejaculates of the males in a replicate were collected individually and pooled JCryoson BV, Cryoson BV, ZH Middenbeemster, The Netherlands. immediately afterwards. The pooled semen was diluted fivefold with the Beltsville Poultry Semen Extender (BPSE, Sexton, 9, 98; Sexton and Fewlass, 98), to which DMSO was added as cryoprotectant. The final concentration of DMSO was.%. Collection and dilution was carried out in the poultry house and was finished within min. The diluted semen was stored at C and transferred to the laboratory for cryopreservation. Cryopreservation The semen was transferred at C to.- ml medium-sized straws that were sealed with polyvinylalcohol. The straws were placed horizontally in a canister for freezing. Freezing started about h after collection of the semen and was carried out in a biological freezer. From to - C the temperature dropped at a rate of C/min; from - to -9 C, the temperature dropped C/min. At -9 C the canister was plunged into liquid nitrogen (-9 C). Thawing and Inseminations Each straw was thawed for min in an alcohol bath at C. The straw was then dried and the sealing was removed. Insemination was intravaginally (about cm deep) immediately from the straw with a rabbit insemination pistol. Each hen received. ml of pooled diluted semen from the straw. All inseminations of one replicate were carried out on the same afternoon; inseminations of the next replicate took place wk later. Determination of Semen Quality The concentration of spermatozoa was determined in the cooled semen spectrophotometrically (X = run). The percentage of live, dead, and abnormal spermatozoa was determined both before cryopreservation and after thawing in semen smears colored with vital staining (Van der Schaaf, 9). After cooling, the smears were made directly from the tube with pooled semen; after thawing, the contents of three straws were mixed to produce the smears. Downloaded from at Wageningen UR Library on September 9,

3 8 VAN VOORST AND LEENSTRA Egg Collection and Determination of Fertility The eggs were collected daily for d, starting at Day after insemination. The eggs were incubated for d. Fertility was determined by candling and by internal examination of clear eggs. After candling the incubation process was terminated. Statistical Analysis Three blocks of individual females were used. In each block six randomly allocated hens received semen of one of the d collection. During the four replicates the hens received semen of the same collection day. The average fertility of the six hens per block was used as experimental unit. A regression analysis was carried out according to the model: InPWd - P ijk. )] = N + bi + R w + DjR k + e, -ijk TK where ln[pij k /(l - P^)] is a binomial; P^ is the percentage fertility in block i, of semen collected at day j, in replicate k; N is the overall level; b s is the effect of block (i =,, ); Dj is the effect of collection of semen at Day,,, or ; R k is the effect of replicate (k =,,,); and e^ is the residual. It should be noted that replicate is confounded with age of both males and females. Differences were considered significant when P <.. The data on semen concentration and of the differential counts were not analyzed statistically. RESULTS AND DISCUSSION Concentration of Spermatozoa The concentration of spermatozoa in undiluted semen is given in Table. In each of the replicate trials the concentration seemed to diminish with day of collection. The average concentration of spermatozoa from the males used in Replicate and seemed to be higher than from the males used in Replicate and. The total number of spermatozoa inseminated varied from 9 million Pay, Replicate ) to million (Day, Replicate ). Differential Counts in Semen Smears The results of the differential counts before and after cryopreservation are given in Table. In diluted, cooled semen at least 9% of the spermatozoa were classified as normal and alive. A maximum of % abnormal spermatozoa (dead and alive combined) was observed. After freezing and thawing, a large proportion of the spermatozoa were dead. In Replicate the percentage of live, normal spermatozoa in the thawed semen seemed to be higher than in the other replicate trials. No clear explanation for this apparent difference can be given. The percentage of abnormal spermatozoa after thawing (dead and alive combined) appeared slightly higher in Replicates and than in Replicates and. There was no indication that the quality of the semen improved over the -d collection. From the sperm concentration and the results of the differential counts, it can be calculated that each hen received at least million live, normal spermatozoa. The mean of inseminated alive and normal spermatozoa for the four replicates of Day,,, and, were respectively, 8,,, and million. The insemination dose of. ml was chosen to create a practical standard procedure. Insemination directly from the straw avoids manipulation of the frozen-thawed semen. Downloaded from at Wageningen UR Library on September 9, TABLE. Mean concentrations of spermatozoa x 9 per milliliter of the ejaculates of males, massaged on consecutive d in four replicates Day Replicate Replicate Replicate Replicate Mean ithe males in Replicate are identical to those in Replicate and those in Replicate to those in Replicate

4 FERTILITY OF FROZEN FOWL SEMEN 9 TABLE. Results of differential counts of vital-stained smears of processed unfrozen and frozen-thawed semen, daily collected on consecutive d over four replicates Replicate Day LN Processed unfrozen L. Duplicate mean of differential counts from vital-stained semen smears Processed frozen-thawed DN DA LN LA DN DA J LN = percentage live and normal spermatozoa; LA = percentage live and abnormal spermatozoa; DN : percentage dead and normal spermatozoa; DA = percentage dead and abnormal spermatozoa. Fertility The percentage of fertile eggs for the -d collection and the four replicates is given in Table. Per day and replicate, the percentage is based on more than eggs. The percentage fertility in Replicate was significantly higher than in Replicates or. The fertility in Replicate was significantly higher than that in Replicate. This seems in accordance with the results from the differential counts after cryopreservation, but the presence of alive and normal spermatozoa do not guarantee fertilizing ability. In previous experiments (Haije, 99), fertile eggs were seldom found more than 9 d after a single insemination with frozenthawed semen. He reported that inseminations twice a week with frozen-thawed semen had a cumulative positive effect on fertility. In this experiment, 8 d after insemination fertility still was high. The weekly inseminations of the same hens consequently might have had a cumulative effect in improving fertility rate, explaining Downloaded from at Wageningen UR Library on September 9, TABLE. Fertilizing ability (percentage fertilized eggs per eggs set) over wk in four replicates of frozen-thawed semen from males massaged on successive d Day Replicate. (9). () 9. () 8. (9) x 8.c () SEM.8 Replicate 8. () 8. (9) 9. () 8. (9) 8.8 bc (8). "^Values with no common superscript: in column or row of means differ significantly on the logitscale (P <.). inumber of eggs. Replicate 8. () 8. (8) 9. (8) 9. () 9." (). Replicate 8. () 8. () 9.9 () 9. () 88.* (). 8.= 8.<: 9.8" 88.b 8.8 X () () () (9) (,) SEM.8.8..

5 VAN VOORST AND LEENSTRA the higher fertility rate in Replicates and than in Replicate. Percentage of fertility of semen collected on Days and was significantly higher than on Days or. Semen collected on Day provided a higher fertility than semen collected on Day. The differential counts, based on the replicates, related very well to the fertility data. However, based on the collection days, a relation between percentage or number of live, normal spermatozoa and fertility was not clear. This might be due to degeneration of spermatozoa that are stored too long in the different ducts. It might be that degeneration or aging of spermatozoa is not reflected in the differential counts, but that fertilizing ability after freezing and thawing of spermatozoa is better indication for degeneration. However, too frequent collection might reduce the concentration of spermatozoa too much. Consequently, an optimum can be expected for frequency of collection. The extremely high fertility of frozenthawed fowl semen in this experiment might have been due to the use of Milli-Q water instead of distilled water used in previous experiments at our Institute (Haije, 99). However, in a direct comparison of diluents based on Milli-Q or distilled water, a high percentage of fertility (> %) of cryopreserved semen was found in both cases (unpublished data). Conclusion The quality of poultry semen for freezing and thawing reached an optimum at the rd d of a series of four daily ejaculations. It is thus advised to select males with a good semen quality and to have them ejaculate for the d before semen is collected for cryopreservation. REFERENCES Bellagamba, F., S. Cerolini, and L. G. Cavalchini, 99. Cryopreservation of poultry semen: a review. World's Poult. Sci. J. 9:-. Burrows, W. H., and J. P. Quinn, 9. A method of obtaining spermatozoa from the domestic fowl. Poultry Sci. :-. Buss, E. G., 99. Cryopreservation of rooster sperm. Poultry Sci. :9-9. De Reviers, M, and J. Williams, 98. Testis development and production of spermatozoa in the cockerel (Gallus domesticus). Pages 8- in: Reproductive Biology of Poultry. Cunningham, F.J., P.E. Lake, and D. Hewitt, ed. British Poultry Science Ltd., Longman Group, Harlow, UK. Froman, D. P., 99. Extra-gonadal maturation of fowl sperm: a genetic model. Pages - in: Control of Fertility in Domestic Birds. Station de Recherches Avicoles, Tours, France. Haije, U., 99. Evaluation of Cryopreservation of Fowl Semen. Ph.D. thesis. University of Utrecht, Centrum voor Onderzoek en Voorlichting voor de Pluimveehouderij. Publication, Beekbergen, The Netherlands. Hammerstedt, R. H., and J. K. Graham, 99. Cryopreservation of poultry sperm: The enigma of glycerol. Cryobiology 9:-8. Lake, P. E., 98. The history and future of the cryopreservation of avian germ plasma. Poultry Sci. :-. Leenstra, F. R., 988. Selection for leanness: Results of the Spelderholt experiment. Pages 9-9 in: Leanness in Domestic Birds: Genetic, Metabolic and Hormonal Aspects. B. Leclercq and C. C. Whitehead, ed. Butterworths, London, England. Quay, W. B., 98. Spontaneous continuous release of spermatozoa and its predawn surge in male passerine birds. Gamete Res. :8-9. Sexton, T. J., 9. A new poultry semen extender.. Effect of extension on the fertility of chicken semen. Poultry Sci. :-. Sexton, T. J., 98. A new poultry semen extender.. Effect of storage conditions on the fertilizing capacity of chicken semen stored at C Poultry Sci. :8-89. Sexton, T. J., 98. Optimal rates for cooling chicken semen from + to -9 C. Poultry Sci. 9: -. Sexton, T. J., and T. A. Fewlass, 98. A new poultry semen extender.. Effect of the diluent components on the fertilizing capacity of chicken semen stored at C. Poultry Sci. :-8. Van der Schaaf, A., 9. Vitaalkleuring van stieren sperma met een oplossing van aniline-blauw en eosine. Tijdschr. Diergeneeskd. :8-88. Downloaded from at Wageningen UR Library on September 9, Millipore Corp., Bedfort, MA.