Fertilizing ability of turkey semen diluted with
simple sugar-based extenders after cooled storage
E. AKÇAY1*, Ö. VARIŞLI1, N. TEKİN1
1
Department of Animal Reproduction and AI, Faculty of Veterinary Medicine, University of
Ankara, 06110 Ankara, Turkey.
*Corresponding author: [email protected]
It is now possible to store turkey semen for 4 to 24 h without appreciable loss in fertility and
hatchability. Optimum conditions for 24 or 48 h storage have not been fully determined.
Development of new storage media, allowing the insemination of very low numbers of
spermatozoa, may provide interesting possibilities regarding the use of superior sires. This study
was conducted to evaluate effects of simple sugar-based extenders (0.3 M Glucose, 0.6 M
Sucrose) or Ringer + glucose solution after 24 h or 48 h of cooled storage on fertility. Standard
bred bronze turkey toms 40 weeks of age were used. Semen was collected by abdominal massage
twice a week throughout 4 weeks of study. Ejaculates were pooled, mixed thoroughly, and
diluted to insemination dose (200 x 106) with 1) 0.3 M glucose, 2) 0.6 M sucrose, and 3) ringer +
glucose solution. Diluted semen were placed in 10 ml erlenmeyer flasks. All flasks were covered
with parafilm and stored in a refrigerator for 24 or 48 h at 4 oC. Changes in sperm motility were
determined before and after holding. Sperm stored in 0.6 M sucrose solution was not used for
the inseminations because of low motility (10 % after 24 h, 0 % after 48 h). Hens were
inseminated two times in the first week and once weekly with 200 x 106 viable spermatozoa for 4
weeks. The mean fertility rates for sperm stored with two extenders (0.3 M glucose and Ringer +
glucose) at 4 oC were: 90.9% ( 24 h, 0.3 M glucose); 79.1% ( 48 h, 0.3 M glucose); 69.8 % (24 h,
Ringer + glucose); 30.9 % (48 h, Ringer + glucose); 93.8 % (control, fresh). No significant
difference in fertility rates were observed between sperm stored in glucose-based extender for 24
hr and the unstored control, but differences between extenders were significant (p<0.05). Thus,
glucose-based extender was beneficial and provided better conditions for cooled storage than did
the others. There was no difference in hatchability of fertile eggs in the treatments. The results
show that turkey semen can be stored in glucose-based extender for 24 or 48 h with a minimal
loss in fertility and hatchability.
Keywords: turkey; cooled semen; extender; motility; fertility
Introduction
Cooled storage of semen is used to reduce metabolism and to maintain sperm viability over an
extended period of time. Excellent sperm survival and fertility are obtained when diluted turkey semen
is stored for 6 h in a cool liquid state before artificial insemination (Sexton and Giesen, 1983).
However, liquid storage beyond 6 h or freezing of semen has not preserved the viability of turkey
semen at the level necessary for commercial use (Sexton, 1988; Thurston, 1995). The improvement of
long-term liquid storage procedures of turkey semen is important since the commercial production of
this bird relies almost entirely on artificial insemination.
Several methods have been described for the cooled storage and cryopreservation of turkey semen.
The influence of the variability in biological material on fertilizing ability of stored semen,
multiplicity of preservation procedures affects the consistency of fertilization results. Differences in
extenders, additives, and environmental conditions make it difficult to get variable estimations of the
efficiency of various procedures. Turkey spermatozoa rapidly lose viability and fertilizing capacity
when stored either undiluted or diluted at physiological temperatures (Leighton et al., 1968; Lake and
Ravie, 1982). In order to maintain the fertilizing ability of in vitro-stored spermatozoa, sperm must be
pre-cooled to 2–8 °C (Giesen and Sexton, 1983; Akcay et al., 1997; Donoghue and Wishart,
2000) and diluted in an appropriate extender (Sexton, 1988; Thurston et al., 1994; Akcay et al., 1997).
Many extenders have been developed for bird semen storage (Lake and Ravie, 1982; Graham et al.,
1982; Sexton, 1988; Morrell et al., 2005); most of these are saline buffers appropriate for the
immediate survival of spermatozoa because they provide osmotic pressure (330–400 mOsm) and pH
(7.0–7.4) identical to those of seminal plasma (Thurston, 1995). Extenders should also contain energy
substrates. Therefore, extenders used for poultry semen are enriched in carbohydrates (glucose or
fructose) and in other components likely to provide energy (citrate, glutamate, acetate) (Graham et al.,
1982; Christensen, 1995; Thurston, 1995). Briefly, most of the extenders provide the requirements for
both energy metabolism and buffering capacity.
It is difficult to maintain semen quality after in vitro liquid storage of turkey semen up to 24 h
(Bakst and Cecil, 1992; Thurston et al., 1994), and the problem is enhanced with animal aging
(Douard et al., 2003). In parallel with decreased fertility, changes in motility (Douard et al., 2000;
Donoghue and Donoghue, 1997) may indicate metabolic disturbances. Maintenance of fertilizing
capacity of poultry sperm for up to 24 and 48 h requires the provision of oxygen, a extender that
supplies fructose or glucose as a substrate for the production of ATP, sufficient buffering capacity to
maintain pH, and an ambient temperature 4-7 oC (Wishart, 1982). In addition, Turkey spermatozoa
differ from fowl spermatozoa in many aspects of their metabolism and resistance to cooled storage.
The metabolism of glucose and acetate are lower in avian than mammalian spermatozoa (Scott et al.,
1962). The exogenous substrates added to extenders for turkey semen may not be sufficient or
appropriate for the energy needs of gametes during in vitro storage.
It is now possible to store turkey semen for 4 to 24 h with a minimal loss in fertility and
hatchability. Optimum conditions for 24 or 48 h storage have not been fully determined. This study
was conducted to evaluate effects of simple carbohydrate-based solutions (0.3 M Glucose, 0.6 M
Sucrose) and ringer solution containing glucose on sperm motility and fertilizing ability after 24 h or
48 h of cooled storage.
Materials and Methods
Standard bred bronze toms 40 weeks of age were used and reared in a temperature regulated
poultry house with artificial lighting (16 h light:8 h dark) and free access to feed and water. The semen
was collected two times a week throughout the 4 weeks of the study by dorso-abdominal massage.
Care was taken to avoid any contamination of semen with cloacal products. Yellow and abnormal
semen samples were also discarded to avoid the deleterious seminal products. The ejaculates of 6 toms
were pooled and in total 12 semen pools were used.
Spermatozoal motility was evaluated using a phase-contrast microscope at x400 magnification and
was expressed as percentage of motile spermatozoa before and after holding. For the evaluation of
motility, about 10 μl drop of the 1:1 diluted semen placed on a microscope slide and covered with a
coverslip. For each sample, at least five microscopic fields were observed. The sperm motility was
observed by two observer. Only samples showing high motility (>80%) were used for cooled storage.
The sperm density (two replicates) was determined using 100 μm deep Thoma haemocytometer (TH100; Hecht-Assistent, Sondheim, Germany) and expressed as spermatozoa x109/ ml. Semen and
extender pH was measured using indicator papers (Merck 5.5-9).
Collected semen from 6 males were pooled in equal amounts according to the required semen
volume and the sperm density to eliminate the effect of individual variability of gamete donors. Three
extenders were tested on split samples of pooled semen. The semen and extenders were kept at 2 °C
prior to dilution. The pooled semen was diluted to insemination dose (200 x 106) with 1) 0.3 M
glucose (300 mOsm, 5.5 pH), 2) 0.6 M sucrose (600 mOsm, 5.0 pH), and 3) ringer solution containing
glucose (9.5 g NaCl, 0.2 g KCl, 0.26 g CaCl2, 0.2 g NaHCO3, 1 g glucose, 1 lt add. distiled water, 350
mOsm, 7.5 pH). Diluted samples were mixed thoroughly and placed in 10 ml erlenmeyer flasks. All
flasks were covered with parafilm and stored in a refrigerator for 24 or 48 h at 4 oC.
The fertilizing ability of spermatozoa was assessed by intravaginal insemination of 50 females per
experiment group and unstored control group. Hens were inseminated two times in the first week and
once weekly with 200 x 106 viable spermatozoa for 4 weeks with fresh semen or semen stored for 24 h
at 4 °C or semen stored for 48 h at 4 °C. Eggs were collected daily, stored in a cold room at 3-5 oC and
75% relative humidity and incubated weekly. The fertility rate (fertile/incubated eggs×100) was
determined by candling 7 days after the start of incubation. Hatching rates (hatching/fertile eggsx100)
was determined by hatching of fertile eggs about 28 days after start of incubation.
The data were subjected to analysis of variance (ANOVA). Duncan’s multiple comparison test was
used for the post-hoc comparisons. Differences were considered significant at the probability level of
0.05.
Results and Discussion
The average rates of spermatozoal motility, fertility and hatchability of eggs by treatment and the
analyses of variance for these data are presented in Table 1. Fertility of unstored semen was the
highest, but differences between unstored control and semen stored with glucose solution for 24 h
were not significant (p>0.05). Stored samples in glucose solution maintained motility and fertilizing
capacity during cooled storage for 24 h and 48 h as unstored control group. On the other hand,
fertilizing capacity of spermatozoa diluted in glucose was different from that of semen diluted in the
other extenders (p<0.05). Fertility of semen stored for 24 h and 48 h was highest stored in glucose
(90.9%, 79.1%) followed by values for storage in sucrose (0%), ringer + glucose (69.8%, 30.9%). No
significant differences were observed for hatchability of fertile eggs due to storing semen in different
extenders (p>0.05).
Table 1. Effect of semen extender on the motility and fertility of turkey sperm stored for 24 h or 48 h at 4 oC and
hatchability of fertile eggs.
Motility
after
dilution
(%)
0.3 M Glucose
Semen stored
Motility
(%)
24 h
Fertility
(%)
Hatchability
(%)
Motility
(%)
48 h
Fertility
(%)
Hatchability
(%)
91.3±2.6
92.5±1.9a
90.9±1.5b
85.1±0.01a
85.5±2.2b
79.1±1.2b
83.2±0.01a
0.6 M Sucrose
50.0±2.0
10.0±0.01c
-
-
-
-
-
Ringer+Glucose
85.5±2.2
80.0±3.2b
69.8±3.6a
84.5±0.01a
50.6±2.6a
30.9±2.4c
83.4±0.01a
90.5±2.9b
93.8±1.5b
82.0±0.01a
90.5±2.2b
91.6±2.9a
84.0±0.01a
1
Extenders
Control (unstored semen)
a,b,c
Means(±sem) in columns with different superscripts are significantly different (p<0.05)
There were four replicates per treatment
1
Although the metabolism of glucose and acetate are lower in avian than mammalian spermatozoa
and glycolytic enzyme activities of turkey sperm are 2–10 times lower than in fowl (Scott et al., 1962;
Wishart, 1982), the higher fertility associated with glucose solution than with sucrose or ringer +
glucose solutions agrees with Graham et al. (1982) who reported that replacement of approximately
half of the extender with an iso-osmolar glucose solution yielded higher percentages of motile
spermatozoa with both fresh and frozen-thawed semen samples. Replacement of glucose with other
carbohydrates did not enhance recovery of frozen-thawed semen. In contrast, turkey hens were
inseminated with either fresh or stored (4 h at 15 oC) semen suspended in a sugar-free medium or one
containing 15 mM glucose or fructose. Rates of fertilization of eggs collected for 15 days after
insemination were not affected by the presence or absence of glucose or fructose in the medium nor by
storage (Pinto et al., 1985).
In the study, three extenders used varied in osmolarity from 300-600 mOsm. Like fowl semen (Van
Wambeke, 1977), turkey spermatozoa can tolerate hypertonic solutions during storage (Huyghebaert
and Groote, 1983). In the experiment of Sexton and Giesen (1982) no loss in fertility was reported
using a 280 mOsm extender. In view of these results it remains difficult to pronounce an optimal
tonicity per se since other physical and chemical characteristics of the extender varied in these
different studies.
The pH of the three extenders in the present study varied from 5.0 to 7.5. The glucose extender at
pH 5.5 gives better fertility results than the others. In contrast, the extender with the lowest pH
(sucrose, pH 5.0) worsened viability, motility and fertility. Besides osmotic pressure of sucrose
solution was the highest (600 mOsm). Therefore, sperm motility might be affected due to hypertonic
media. Giesen and Sexton (1983) showed that constant agitation of turkey semen stored in a pH 7.5
extender (BPSE) for 18 h at 5 oC resulted in significantly lower fertility than semen stored in a pH 6.5
extender (BTSE) for 18 h at 5 oC with or without intermittent agitation. Similar results were reported
by Lake at al. (1984). The present study found that spermatozoa stored in glucose solution (24 and 48
h at 4 oC, no agitation) showed better motility and fertility than spermatozoa stored in sucrose solution
or ringer + glucose solution. Therefore, the pH of the extender and agitation, which is used to aerate
the semen, may have created bad conditions for the spermatozoa during storage. Giesen and Sexton
(1983) also noted that holding turkey semen for 18 h at 5 oC did not affect fertility but that fertility was
significantly depressed when semen was held for 18 h at 15, 25, or 36 oC. They suggested that storage
temperatures greater than 15 oC may make the sperm plasma membrane more labile.
The reports of Sexton (1977), Donoghue et al. (1995) confirm the fact that sperm number in excess
of 50 million and less than 200 million will assure consistently high fertility levels. Sexton (1987)
reported that fertility of stored turkey semen diluted 1:2 was lower that of semen diluted 1:1 and 2:1.
In earlier studies Sexton (1976) showed that diluting semen at high rates had a negative influence on
spermatozoa and when diluted above a rate of 1:4, metabolic activity increased and fertilizing ability
decreased. When spermatozoa were maintained in the diluted state and then reconcentrated, fertility
was significantly depressed with semen from the high dilution (1:8, 1:12) levels. Cecil and Bakst
(1993) reported that formation of MAL per spermatozoon was dependent on, and inversely correlated
with, the concentration of spermatozoa in the incubation mixture. Therefore, one could conclude the
greater the dilution rate, the greater the rate of lipid peroxidation of spermatozoal membranes
(deleterious dilution effect). According to these researchers, the effect of semen dilution on turkey
spermatozoa is similar to that reported for a number of mammalian species. In the present study,
semen was diluted to insemination dose (approximately 1:25). But it was not observed deleterious
dilution effect or low fertility. The results of the present study suggested that glucose extender and
high dilution rates may be useful for the cooled storage of turkey semen at 4 oC for 24 or 48 h.
These preliminary findings indicated the efficacy of using a simple glucose-based extender during
cooled storage of turkey semen and turkey semen can be stored in glucose-based extender for 24 or 48
h at 4 oC with a minimal loss in fertility. The simplified extender composition, high levels of dilution
and rapid holding time resulted in less complicated storing procedures. But further research is needed
on the effect of sugars, particularly the effect of glucose at molecular level, before recommendations
can be made.
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