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Fertility Rate of Daily Collected and Cryopreserved Fowl Semen
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 15, 7360 AA Beekbergen, The Netherlands
1995 Poultry Science 74:136-140
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, 1993). Cryopreservation of domestic fowl semen results in a decreased fertility rate when compared with fresh, unfrozen semen (Lake, 1985; Bellagamba et al., 1993). Hammerstedt and Graham (1992) summarized recent research on fertility of frozen-thawed fowl semen. They found a fertility of 55% using dimethylsulfoxide (DMSO) and 60% using glycerol. The freezing and thawing protocol using DMSO developed by Sexton (1980) allows for insemination directly after thawing
Received for publication April 5, 1994. Accepted for publication September 22, 1994.
from straws used for storage (Haije, 1990). 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 (1990) 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 5 d. Fewer degenerated spermatozoa are found in semen of males ejaculated relatively frequently (de Reviers and Williams, 1984). In male passerine birds, Quay (1987) 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 (1990) found that daily collection of semen over a period of 12 d did not reduce volume and concentration of the ejaculate nor the quality of the spermatozoa.
136
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
ABSTRACT Semen was collected for 4 consecutive d individually from experimental broiler breeder males that had not been massaged for 7 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 1 wk after a single insemination with the cryopreserved semen was determined. The overall fertility rate in this experiment (83 to 93%) was high, compared with the generally reported fertility rate of frozen-thawed fowl semen. The fertility rate of the semen collected on Days 3 and 4 was significantly higher than that of semen collected on Day 1 or 2, indicating that frequent collection over the 4-d period enhances the fertility rate of the semen. (Key words: fowl, semen, cryopreservation, fertility rate)
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.
immediately afterwards. The pooled semen was diluted fivefold with the Beltsville Poultry Semen Extender (BPSE, Sexton, 1977, 1978; Sexton and Fewlass, 1978), to which DMSO was added as cryoprotectant. The final concentration of DMSO was 4.5%. Collection and dilution was carried out in the poultry house and was finished within 10 min. The diluted semen was stored at 5 C and transferred to the laboratory for cryopreservation. Cryopreservation
The semen was transferred at 5 C to .5The experiment consisted of four repli- mL medium-sized straws that were sealed cates, carried out during 4 consecutive wk. with polyvinylalcohol. The straws were Ten males were used in Replicates 1 and 3 placed horizontally in a canister for freezand 10 different males in Replicates 2 and ing. Freezing started about 1 h after collec4. In each replicate trial, semen was tion of the semen and was carried out in a collected for 4 consecutive d from the biological freezer.1 From 5 to -20 C the same 10 males. temperature dropped at a rate of 1 C/min; from -20 to -95 C, the temperature dropped Chickens 25 C/min. At -95 C the canister was plunged into liquid nitrogen (-196 C). Twenty broiler breeder males of a Spelderholt selection line (FC line, Leenstra, 1988) were used. The males were 39 wk of Thawing and Inseminations age at the start of the experiment. SeventyEach straw was thawed for 2 min in an two White Leghorn females were insemialcohol bath at 5 C. The straw was then nated to determine fertility rate. They were dried and the sealing was removed. Insemi32 wk of age at the start of the experiment. All birds were housed in individual cages. nation was intravaginally (about 6 cm deep) Daily lighting was 14 h light and 10 h immediately from the straw with a rabbit darkness. All the birds received a standard insemination pistol. Each hen received .5 layer diet (16.1% crude protein and 2,850 mL of pooled diluted semen from the straw. kcal ME/kg). The females had feed and All inseminations of one replicate were water available for ad libitum consumption, carried out on the same afternoon; insemithe males were restricted to a feed intake of nations of the next replicate took place 1 wk 125 g/d and had water available during 3 later. h/d. Determination of Semen Quality Collection and Dilution of Semen
The concentration of spermatozoa was determined in the cooled semen spectrophotometrically (X = 625 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, 1952). After cooling, the smears were made directly from the tube with pooled semen; after thawing, the contents JCryoson BV6, Cryoson BV, 1462 ZH Middenbeem- of three straws were mixed to produce the smears. ster, The Netherlands.
Semen was collected according to the massage technique of Burrows and Quinn (1935). At the start of each replicate trial the males had not been massaged for 7 d. The semen was collected after a single massage. The ejaculates of the 10 males in a replicate were collected individually and pooled
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
MATERIALS AND METHODS
137
138
VAN VOORST AND LEENSTRA
Egg Collection and Determination of Fertility The eggs were collected daily for 7 d, starting at Day 2 after insemination. The eggs were incubated for 7 d. Fertility was determined by candling and by internal examination of clear eggs. After candling the incubation process was terminated. Statistical Analysis
I n P W d - Pijk ijk.)] = N + bi + Rw + DjRk + 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 = 1,2, 3); Dj is the effect of collection of semen at Day 1,2,3, or 4; Rk is the effect of replicate (k = 1,2,3,4); 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 < .05. 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 1. In each
Differential Counts in Semen Smears The results of the differential counts before and after cryopreservation are given in Table 2. In diluted, cooled semen at least 90% of the spermatozoa were classified as normal and alive. A maximum of 7% abnormal spermatozoa (dead and alive combined) was observed. After freezing and thawing, a large proportion of the spermatozoa were dead. In Replicate 3 the percentage of live, normal spermatozoa in the thawed semen seemed to be higher than in the other replicate trials. N o 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 1 and 2 than in Replicates 3 and 4. There was no indication that the quality of the semen improved over the 4-d collection. From the sperm concentration and the results of the differential counts, it can be calculated that each hen received at least 100 million live, normal spermatozoa. The mean of inseminated alive and normal spermatozoa for the four replicates of Day 1, 2, 3, and 4, were respectively, 168, 150, 154, and 157 million. The insemination dose of .5 mL was chosen to create a practical standard procedure. Insemination directly from the straw avoids manipulation of the frozen-thawed semen.
TABLE 1. Mean concentrations of spermatozoa x 109 per milliliter of the ejaculates of 10 males, massaged on 4 consecutive d in four replicates1 Day
Replicate 1
Replicate 2
Replicate 3
Replicate 4
1 2 3 4 Mean
6.92 6.60 5.85 5.92 6.32
6.42 5.42 4.85 4.78 5.37
5.78 5.78 5.92 5.60 5.77
5.68 4.78 4.60 4.25 4.83
6.20 5.64 5.30 5.14 5.57
iThe males in Replicate 1 are identical to those in Replicate 3 and those in Replicate 2 to those in Replicate 4.
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
Three blocks of 24 individual females were used. In each block six randomly allocated hens received semen of one of the 4 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:
of the replicate trials the concentration seemed to diminish with day of collection. The average concentration of spermatozoa from the males used in Replicate 1 and 3 seemed to be higher than from the males used in Replicate 2 and 4. The total number of spermatozoa inseminated varied from 692 million P a y 1, Replicate 1) to 425 million (Day 4, Replicate 4).
139
FERTILITY OF FROZEN FOWL SEMEN TABLE 2. Results of differential counts of vital-stained smears of processed unfrozen and frozen-thawed semen, daily collected on 4 consecutive d over four replicates Duplicate mean of differential counts from vital-stained semen smears1 Processed unfrozen Day
LN
L.
1
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
94 93 90 91 91 91 92 91 93 93 90 93 91 91 91 90
4 5 5 5 7 5 5 6 4 5 5 4 5 4 5 4
2
3
4
DN
DA
LN
LA
DN
DA
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
23 21 23 26 22 23 25 28 39 34 37 39 26 29 31 29
8 9 6 7 9 7 6 7
67 66 68 64 66 69 65 63 53 58 56 56 68 63 63 63
2 4 3 3 3 1 4 2 3 2 2 1 2 3 1 3
5 6 5 4 4 5 5 5
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.
spermatozoa do not guarantee fertilizing ability. The percentage of fertile eggs for the In previous experiments (Haije, 1990), 4-d collection and the four replicates is fertile eggs were seldom found more than 9 given in Table 3. Per day and replicate, the d after a single insemination with frozenpercentage is based on more than 100 eggs. thawed semen. He reported that inseminaThe percentage fertility in Replicate 3 was tions twice a week with frozen-thawed significantly higher than in Replicates 1 or semen had a cumulative positive effect on 2. The fertility in Replicate 4 was signifi- fertility. In this experiment, 8 d after cantly higher than that in Replicate 1. This insemination fertility still was high. The seems in accordance with the results from weekly inseminations of the same hens the differential counts after cryopreserva- consequently might have had a cumulative tion, but the presence of alive and normal effect in improving fertility rate, explaining Fertility
TABLE 3. Fertilizing ability (percentage fertilized eggs per eggs set) over 1 wk in four replicates of frozen-thawed semen from males massaged on 4 successive d Day 1 2 3 4 x SEM
Replicate 1 76.1 75.7 94.0 82.6 82.2c 1.8
(109)1 (111) (116) (109) (445)
Replicate 2
Replicate 3
Replicate 4
SEM
X
82.7 83.5 90.0 87.2
(110) (109) (110) (109)
87.5 86.1 94.4 93.6
(104) (108) (108) (110)
84.2 87.7 92.9 90.1
(114) (106) (113) (111)
82.6= 83.2<: 92.8" 88.4b
85.8bc 1.6
(438)
90.5" 1.4
(430)
88.7* 1.5
(444)
86.8
(437) (434) (447) (439)
1.8 1.8 1.2 1.5
(1,757)
"^Values with no common superscript:in column or row of means differ significantly on the logitscale (P < .05). iNumber of eggs.
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
Replicate
Processed frozen-thawed
140
VAN VOORST AND LEENSTRA
Conclusion The quality of poultry semen for freezing and thawing reached an optimum at the 3rd 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 2 d before semen is collected for cryopreservation.
2
Millipore Corp., Bedfort, MA 01730.
REFERENCES Bellagamba, F., S. Cerolini, and L. G. Cavalchini, 1993. Cryopreservation of poultry semen: a review. World's Poult. Sci. J. 49:157-166. Burrows, W. H., and J. P. Quinn, 1935. A method of obtaining spermatozoa from the domestic fowl. Poultry Sci. 14:251-254. Buss, E. G., 1993. Cryopreservation of rooster sperm. Poultry Sci. 72:944-954. De Reviers, M, and J. Williams, 1984. Testis development and production of spermatozoa in the cockerel (Gallus domesticus). Pages 183-202 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., 1990. Extra-gonadal maturation of fowl sperm: a genetic model. Pages 41-45 in: Control of Fertility in Domestic Birds. Station de Recherches Avicoles, Tours, France. Haije, U., 1990. Evaluation of Cryopreservation of Fowl Semen. Ph.D. thesis. University of Utrecht, Centrum voor Onderzoek en Voorlichting voor de Pluimveehouderij. Publication 532, Beekbergen, The Netherlands. Hammerstedt, R. H., and J. K. Graham, 1992. Cryopreservation of poultry sperm: The enigma of glycerol. Cryobiology 29:26-38. Lake, P. E., 1985. The history and future of the cryopreservation of avian germ plasma. Poultry Sci. 65:1-15. Leenstra, F. R., 1988. Selection for leanness: Results of the Spelderholt experiment. Pages 59-69 in: Leanness in Domestic Birds: Genetic, Metabolic and Hormonal Aspects. B. Leclercq and C. C. Whitehead, ed. Butterworths, London, England. Quay, W. B., 1987. Spontaneous continuous release of spermatozoa and its predawn surge in male passerine birds. Gamete Res. 16:83-92. Sexton, T. J., 1977. A new poultry semen extender. 1. Effect of extension on the fertility of chicken semen. Poultry Sci. 56:1443-1446. Sexton, T. J., 1978. A new poultry semen extender. 3. Effect of storage conditions on the fertilizing capacity of chicken semen stored at 5°C Poultry Sci. 57:285-289. Sexton, T. J., 1980. Optimal rates for cooling chicken semen from +5 to -196 C. Poultry Sci. 59: 2765-2770. Sexton, T. J., and T. A. Fewlass, 1978. A new poultry semen extender. 2. Effect of the diluent components on the fertilizing capacity of chicken semen stored at 5°C. Poultry Sci. 57:277-284. Van der Schaaf, A., 1952. Vitaalkleuring van stieren sperma met een oplossing van aniline-blauw en eosine. Tijdschr. Diergeneeskd. 77:815-818.
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
the higher fertility rate in Replicates 3 and 4 than in Replicate 1. Percentage of fertility of semen collected on Days 3 and 4 was significantly higher than on Days 1 or 2. Semen collected on Day 3 provided a higher fertility than semen collected on Day 4. 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-Q2 water instead of distilled water used in previous experiments at our Institute (Haije, 1990). However, in a direct comparison of diluents based on Milli-Q or distilled water, a high percentage of fertility (> 70%) of cryopreserved semen was found in both cases (unpublished data).
1995 Poultry Science 74:136-140
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, 1993). Cryopreservation of domestic fowl semen results in a decreased fertility rate when compared with fresh, unfrozen semen (Lake, 1985; Bellagamba et al., 1993). Hammerstedt and Graham (1992) summarized recent research on fertility of frozen-thawed fowl semen. They found a fertility of 55% using dimethylsulfoxide (DMSO) and 60% using glycerol. The freezing and thawing protocol using DMSO developed by Sexton (1980) allows for insemination directly after thawing
Received for publication April 5, 1994. Accepted for publication September 22, 1994.
from straws used for storage (Haije, 1990). 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 (1990) 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 5 d. Fewer degenerated spermatozoa are found in semen of males ejaculated relatively frequently (de Reviers and Williams, 1984). In male passerine birds, Quay (1987) 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 (1990) found that daily collection of semen over a period of 12 d did not reduce volume and concentration of the ejaculate nor the quality of the spermatozoa.
136
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
ABSTRACT Semen was collected for 4 consecutive d individually from experimental broiler breeder males that had not been massaged for 7 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 1 wk after a single insemination with the cryopreserved semen was determined. The overall fertility rate in this experiment (83 to 93%) was high, compared with the generally reported fertility rate of frozen-thawed fowl semen. The fertility rate of the semen collected on Days 3 and 4 was significantly higher than that of semen collected on Day 1 or 2, indicating that frequent collection over the 4-d period enhances the fertility rate of the semen. (Key words: fowl, semen, cryopreservation, fertility rate)
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.
immediately afterwards. The pooled semen was diluted fivefold with the Beltsville Poultry Semen Extender (BPSE, Sexton, 1977, 1978; Sexton and Fewlass, 1978), to which DMSO was added as cryoprotectant. The final concentration of DMSO was 4.5%. Collection and dilution was carried out in the poultry house and was finished within 10 min. The diluted semen was stored at 5 C and transferred to the laboratory for cryopreservation. Cryopreservation
The semen was transferred at 5 C to .5The experiment consisted of four repli- mL medium-sized straws that were sealed cates, carried out during 4 consecutive wk. with polyvinylalcohol. The straws were Ten males were used in Replicates 1 and 3 placed horizontally in a canister for freezand 10 different males in Replicates 2 and ing. Freezing started about 1 h after collec4. In each replicate trial, semen was tion of the semen and was carried out in a collected for 4 consecutive d from the biological freezer.1 From 5 to -20 C the same 10 males. temperature dropped at a rate of 1 C/min; from -20 to -95 C, the temperature dropped Chickens 25 C/min. At -95 C the canister was plunged into liquid nitrogen (-196 C). Twenty broiler breeder males of a Spelderholt selection line (FC line, Leenstra, 1988) were used. The males were 39 wk of Thawing and Inseminations age at the start of the experiment. SeventyEach straw was thawed for 2 min in an two White Leghorn females were insemialcohol bath at 5 C. The straw was then nated to determine fertility rate. They were dried and the sealing was removed. Insemi32 wk of age at the start of the experiment. All birds were housed in individual cages. nation was intravaginally (about 6 cm deep) Daily lighting was 14 h light and 10 h immediately from the straw with a rabbit darkness. All the birds received a standard insemination pistol. Each hen received .5 layer diet (16.1% crude protein and 2,850 mL of pooled diluted semen from the straw. kcal ME/kg). The females had feed and All inseminations of one replicate were water available for ad libitum consumption, carried out on the same afternoon; insemithe males were restricted to a feed intake of nations of the next replicate took place 1 wk 125 g/d and had water available during 3 later. h/d. Determination of Semen Quality Collection and Dilution of Semen
The concentration of spermatozoa was determined in the cooled semen spectrophotometrically (X = 625 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, 1952). After cooling, the smears were made directly from the tube with pooled semen; after thawing, the contents JCryoson BV6, Cryoson BV, 1462 ZH Middenbeem- of three straws were mixed to produce the smears. ster, The Netherlands.
Semen was collected according to the massage technique of Burrows and Quinn (1935). At the start of each replicate trial the males had not been massaged for 7 d. The semen was collected after a single massage. The ejaculates of the 10 males in a replicate were collected individually and pooled
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
MATERIALS AND METHODS
137
138
VAN VOORST AND LEENSTRA
Egg Collection and Determination of Fertility The eggs were collected daily for 7 d, starting at Day 2 after insemination. The eggs were incubated for 7 d. Fertility was determined by candling and by internal examination of clear eggs. After candling the incubation process was terminated. Statistical Analysis
I n P W d - Pijk ijk.)] = N + bi + Rw + DjRk + 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 = 1,2, 3); Dj is the effect of collection of semen at Day 1,2,3, or 4; Rk is the effect of replicate (k = 1,2,3,4); 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 < .05. 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 1. In each
Differential Counts in Semen Smears The results of the differential counts before and after cryopreservation are given in Table 2. In diluted, cooled semen at least 90% of the spermatozoa were classified as normal and alive. A maximum of 7% abnormal spermatozoa (dead and alive combined) was observed. After freezing and thawing, a large proportion of the spermatozoa were dead. In Replicate 3 the percentage of live, normal spermatozoa in the thawed semen seemed to be higher than in the other replicate trials. N o 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 1 and 2 than in Replicates 3 and 4. There was no indication that the quality of the semen improved over the 4-d collection. From the sperm concentration and the results of the differential counts, it can be calculated that each hen received at least 100 million live, normal spermatozoa. The mean of inseminated alive and normal spermatozoa for the four replicates of Day 1, 2, 3, and 4, were respectively, 168, 150, 154, and 157 million. The insemination dose of .5 mL was chosen to create a practical standard procedure. Insemination directly from the straw avoids manipulation of the frozen-thawed semen.
TABLE 1. Mean concentrations of spermatozoa x 109 per milliliter of the ejaculates of 10 males, massaged on 4 consecutive d in four replicates1 Day
Replicate 1
Replicate 2
Replicate 3
Replicate 4
1 2 3 4 Mean
6.92 6.60 5.85 5.92 6.32
6.42 5.42 4.85 4.78 5.37
5.78 5.78 5.92 5.60 5.77
5.68 4.78 4.60 4.25 4.83
6.20 5.64 5.30 5.14 5.57
iThe males in Replicate 1 are identical to those in Replicate 3 and those in Replicate 2 to those in Replicate 4.
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
Three blocks of 24 individual females were used. In each block six randomly allocated hens received semen of one of the 4 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:
of the replicate trials the concentration seemed to diminish with day of collection. The average concentration of spermatozoa from the males used in Replicate 1 and 3 seemed to be higher than from the males used in Replicate 2 and 4. The total number of spermatozoa inseminated varied from 692 million P a y 1, Replicate 1) to 425 million (Day 4, Replicate 4).
139
FERTILITY OF FROZEN FOWL SEMEN TABLE 2. Results of differential counts of vital-stained smears of processed unfrozen and frozen-thawed semen, daily collected on 4 consecutive d over four replicates Duplicate mean of differential counts from vital-stained semen smears1 Processed unfrozen Day
LN
L.
1
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
94 93 90 91 91 91 92 91 93 93 90 93 91 91 91 90
4 5 5 5 7 5 5 6 4 5 5 4 5 4 5 4
2
3
4
DN
DA
LN
LA
DN
DA
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
23 21 23 26 22 23 25 28 39 34 37 39 26 29 31 29
8 9 6 7 9 7 6 7
67 66 68 64 66 69 65 63 53 58 56 56 68 63 63 63
2 4 3 3 3 1 4 2 3 2 2 1 2 3 1 3
5 6 5 4 4 5 5 5
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.
spermatozoa do not guarantee fertilizing ability. The percentage of fertile eggs for the In previous experiments (Haije, 1990), 4-d collection and the four replicates is fertile eggs were seldom found more than 9 given in Table 3. Per day and replicate, the d after a single insemination with frozenpercentage is based on more than 100 eggs. thawed semen. He reported that inseminaThe percentage fertility in Replicate 3 was tions twice a week with frozen-thawed significantly higher than in Replicates 1 or semen had a cumulative positive effect on 2. The fertility in Replicate 4 was signifi- fertility. In this experiment, 8 d after cantly higher than that in Replicate 1. This insemination fertility still was high. The seems in accordance with the results from weekly inseminations of the same hens the differential counts after cryopreserva- consequently might have had a cumulative tion, but the presence of alive and normal effect in improving fertility rate, explaining Fertility
TABLE 3. Fertilizing ability (percentage fertilized eggs per eggs set) over 1 wk in four replicates of frozen-thawed semen from males massaged on 4 successive d Day 1 2 3 4 x SEM
Replicate 1 76.1 75.7 94.0 82.6 82.2c 1.8
(109)1 (111) (116) (109) (445)
Replicate 2
Replicate 3
Replicate 4
SEM
X
82.7 83.5 90.0 87.2
(110) (109) (110) (109)
87.5 86.1 94.4 93.6
(104) (108) (108) (110)
84.2 87.7 92.9 90.1
(114) (106) (113) (111)
82.6= 83.2<: 92.8" 88.4b
85.8bc 1.6
(438)
90.5" 1.4
(430)
88.7* 1.5
(444)
86.8
(437) (434) (447) (439)
1.8 1.8 1.2 1.5
(1,757)
"^Values with no common superscript:in column or row of means differ significantly on the logitscale (P < .05). iNumber of eggs.
Downloaded from http://ps.oxfordjournals.org/ at University of Rhode Island on April 5, 2015
Replicate
Processed frozen-thawed
140
VAN VOORST AND LEENSTRA
Conclusion The quality of poultry semen for freezing and thawing reached an optimum at the 3rd 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 2 d before semen is collected for cryopreservation.
2
Millipore Corp., Bedfort, MA 01730.
REFERENCES Bellagamba, F., S. Cerolini, and L. G. Cavalchini, 1993. Cryopreservation of poultry semen: a review. World's Poult. Sci. J. 49:157-166. Burrows, W. H., and J. P. Quinn, 1935. A method of obtaining spermatozoa from the domestic fowl. Poultry Sci. 14:251-254. Buss, E. G., 1993. Cryopreservation of rooster sperm. Poultry Sci. 72:944-954. De Reviers, M, and J. Williams, 1984. Testis development and production of spermatozoa in the cockerel (Gallus domesticus). Pages 183-202 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., 1990. Extra-gonadal maturation of fowl sperm: a genetic model. Pages 41-45 in: Control of Fertility in Domestic Birds. Station de Recherches Avicoles, Tours, France. Haije, U., 1990. Evaluation of Cryopreservation of Fowl Semen. Ph.D. thesis. University of Utrecht, Centrum voor Onderzoek en Voorlichting voor de Pluimveehouderij. Publication 532, Beekbergen, The Netherlands. Hammerstedt, R. H., and J. K. Graham, 1992. Cryopreservation of poultry sperm: The enigma of glycerol. Cryobiology 29:26-38. Lake, P. E., 1985. The history and future of the cryopreservation of avian germ plasma. Poultry Sci. 65:1-15. Leenstra, F. R., 1988. Selection for leanness: Results of the Spelderholt experiment. Pages 59-69 in: Leanness in Domestic Birds: Genetic, Metabolic and Hormonal Aspects. B. Leclercq and C. C. Whitehead, ed. Butterworths, London, England. Quay, W. B., 1987. Spontaneous continuous release of spermatozoa and its predawn surge in male passerine birds. Gamete Res. 16:83-92. Sexton, T. J., 1977. A new poultry semen extender. 1. Effect of extension on the fertility of chicken semen. Poultry Sci. 56:1443-1446. Sexton, T. J., 1978. A new poultry semen extender. 3. Effect of storage conditions on the fertilizing capacity of chicken semen stored at 5°C Poultry Sci. 57:285-289. Sexton, T. J., 1980. Optimal rates for cooling chicken semen from +5 to -196 C. Poultry Sci. 59: 2765-2770. Sexton, T. J., and T. A. Fewlass, 1978. A new poultry semen extender. 2. Effect of the diluent components on the fertilizing capacity of chicken semen stored at 5°C. Poultry Sci. 57:277-284. Van der Schaaf, A., 1952. Vitaalkleuring van stieren sperma met een oplossing van aniline-blauw en eosine. Tijdschr. Diergeneeskd. 77:815-818.
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the higher fertility rate in Replicates 3 and 4 than in Replicate 1. Percentage of fertility of semen collected on Days 3 and 4 was significantly higher than on Days 1 or 2. Semen collected on Day 3 provided a higher fertility than semen collected on Day 4. 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-Q2 water instead of distilled water used in previous experiments at our Institute (Haije, 1990). However, in a direct comparison of diluents based on Milli-Q or distilled water, a high percentage of fertility (> 70%) of cryopreserved semen was found in both cases (unpublished data).