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Beltsville Poultry Semen Extender
Beltsville Poultry Semen Extender. 6. Holding Turkey Semen for Six Hours at 15 C1 T. J. SEXTON US Department of Agriculture, SE-ARS, Avian Physiology Laboratory, Beltsville, Maryland 20705 A. F. GIESEN Department of Poultry Science, Alabama Agricultural Experiment Station, Auburn University, Alabama 36849 (Received for publication August 24, 1981)
1982 Poultry Science 61:1202-1208
INTRODUCTION Artificial insemination (AI) costs t h e US t u r k e y breeding industry a p p r o x i m a t e l y 12 million dollars annually. Semen e x t e n d e r s have been used t o lower the cost of AI b y allowing diluted semen t o be inseminated into m o r e hens t h a n is possible with u n d i l u t e d semen. T h e savings in p r o d u c t i o n costs o b t a i n e d by using e x t e n d e r s is realized because fewer t o m s are required. Additional cost savings could c o m e from short-term holding (6 t o 8 hr) of semen prior t o AI with reduced labor costs and increased p r o d u c t i o n efficiency. Advantages would include t h e t r a n s p o r t of semen over short distances, the establishment of breeder torn farms, b e t t e r utilization of t h e AI labor force, a n d t h e insemination of h e n s late in t h e a f t e r n o o n , a time f o u n d to be m o s t desirable (Johnston, 1977).
1 Scientific Article No. 12-81-10083 of the Alabama Agricultural Experiment Station.
F e w investigations have been c o n d u c t e d on the dilution and storage of t u r k e y semen. Results of past w o r k e r s have indicated t h a t the fertilizing capacity of diluted t u r k e y semen decreased rapidly after 1 or 2 hr storage in vitro (Wilcox and Shaffner, I 9 6 0 ; G r a h a m and Brown, 1 9 7 1 ; Litjen, 1 9 7 2 ) . However, a recent r e p o r t b y Lake ( 1 9 7 8 ) indicated t h a t satisfact o r y fertility levels of 85 t o 8 8 % can be obtained from a single insemination of diluted t u r k e y semen held 5 h r before AI. Preliminary data^ from this l a b o r a t o r y indicated t h a t reasonably good fertility (73%) can be obtained from weekly inseminations with turkey semen diluted in Beltsville P o u l t r y Semen E x t e n d e r (BPSE) and held 6 hr before AI (Sexton, unpublished d a t a ) . T h e primary p u r p o s e of this e x p e r i m e n t was t o measure t h e fertilizing capacity of t u r k e y semen diluted in t h e original BPSE and in three modifications (pH and osmolarity) of BPSE. T h e semen was held for 0 or 6 hr before AI. A secondary objective was t o d e t e r m i n e t h e influence of agitating diluted semen, during holding at 15 C.
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ABSTRACT A series of experiments were conducted to measure the effects of various combinations of extender pH (6.5 vs. 7.5), osmolarity (280 us. 355 mOsmols), and sample agitation on the fertilizing capacity of turkey semen held for 6 hr at 15 C. Respiration of turkey sperm diluted in Beltsville Poultry Semen Extender (BPSE) to pH 6.0 and 280 mOsmols was lower than that of sperm diluted in BPSE with pH 7.5 and 280 mOsmols (mOsm). Sperm respiration was unaffected by extender pH adjusted to 350 mOsm, regardless of pH. Motility of both unstored and stored sperm was significantly lower in BPSE at pH 6.5 than at pH 7.5. The fertilizing capacity of sperm diluted and stored in pH 6.5 extenders did not differ from the fertilizing capacity of the unstored controls. The fertility of unstored sperm diluted in the pH 7.5 extenders did not differ from the controls, but holding for 6 hr at 15 C resulted in lower fertility. Extender osmolarity in the range studied had no influence on sperm motility, respiration, or fertility. Phosphoric acid, when used to adjust pH from 7.5 to 6.5, had no effect on fertility, whereas acetic and hydrochloric acid lowered the fertilizing capacity of stored semen. Sample agitation was critical for maintenance of sperm fertility in samples diluted and stored in pH 7.5 extenders but had no effect on sperm diluted in pH 6.5 extenders. (Key words: diluent, turkey, pH, osmolarity, storage, semen)
BELTSVILLE POULTRY SEMEN EXTENDER TABLE 1. Effect of extender pH and osmolality on oxygen uptake (02) of turkey semen incubated at 15 C for 6 hr (Experiment 1)
pH
mOsm
Accumulative 0 2 uptake/10 9 spermatozoa 1
7.5 7.5 6.5 6.5
355 280 280 355
26.1ac 29.9 a 23.7bc 26.5 a c
Modified BPSE
SEM
2.0
1
Mean and SEM of 6 determinations.
MATERIALS AND METHODS In E x p e r i m e n t s 1 t h r o u g h 4 , second season, nonforce-molted males (Large White) and first season females (Small White) of commercial strains were used, while in E x p e r i m e n t 5 first season males (Large White) a n d first season females (Large White) were used. All five experim e n t s were c o n d u c t e d sequentially. Semen samples were collected from a t least 10 males, pooled immediately after collection, and diluted 1 p a r t semen t o 1 part of e x t e n d e r . All semen samples were diluted within 10 min of semen collection. T h e c o m p o s i t i o n of BPSE for t u r k e y s has been outlined by Sexton ( 1 9 8 0 ) . T h e p H and osmolarity changes were t h e only modifications m a d e t o t h e original BPSE, which has a p H of 7.5 and osmolarity of 355 (mOsm/1 H 2 0 ) . T h e BPSE p H was adjusted from 7.5 t o 6.5 in t w o modified versions by the addition of concentrated HC1 (3 to 4 ml/liter of BPSE), while osmolarity was adjusted t o either 355 or 2 8 0 m O s m b y t h e addition of 9 0 0 or 1300 ml of water, respectively. Regardless of the a m o u n t of water used t o adjust osmolarity, t h e ratio of c o m p o n e n t s in each of t h e modified versions was t h e same. D e t e r m i n a t i o n s of p H a n d o s m o larity were m a d e with a Corning Model 110 p H m e t e r and an O s m e t t e A a u t o m a t i c o s m o m e t e r . T h e pH-osmolarity combinations tested are listed in Table 1. Experiments 1 to 3. T h r e e i n d e p e n d e n t e x p e r i m e n t s were designed t o assess t h e effects of dilution of t u r k e y semen in regular and modified versions of BPSE and held at 15 C for 6 h. In E x p e r i m e n t 1, sperm respiration, sperm
motility, egg fertility, and hatchability were measured. Cumulative sperm respiration (jA 0 2 u p t a k e / 1 0 9 sperm) of diluted t u r k e y sperm over a 6 hr period was d e t e r m i n e d m a n o metrically, hourly for 6 hr, with a Gilson Refrigerated Differential Respirometer, Model GR14. Diluted semen (.5 cc containing a p p r o x i m a t e l y 1.5 X 1 0 9 sperm) was placed in t h e main chamber of a 7 ml capacity i n c u b a t i o n flask within 30 min of semen collection. T h e center well of each flask contained .2 cc K O H (20% w/v) t o absorb t h e C 0 2 p r o d u c e d during incubation. Flasks were equilibrated at 15 C for 10 m i n , t h e n i n c u b a t e d with c o n s t a n t agitation at 8 0 cycle/min. E x p e r i m e n t 2 was designed t o d e t e r m i n e t h e effect of e x t e n d e r pH-osmolarity o n t h e motility of t u r k e y sperm held 6 hr at 15 C. T w o cubic centimeters of semen diluted in one of four e x t e n d e r s were placed in a 10 cc flask, covered lightly with a l u m i n u m foil, and held at 15 C for 6 hr. At 0 and 6 hr, flask c o n t e n t s were mixed, an 5 All aliquot was removed, and progressive motility was estimated on a scale of 0 to 100, microscopically (phase-contrast*) at 4 3 0 x on a microscope slide m a i n t a i n e d at r o o m temperature. In E x p e r i m e n t 3, 4 cc of u n d i l u t e d semen was divided into 4 equal parts, diluted with t h e a p p r o p r i a t e e x t e n d e r , and held at 15 C as described for E x p e r i m e n t 2. One p a r t was used immediately t o inseminate t h e c o n t r o l hens (0 hr) and t h e o t h e r part was held for 6 hr. Eighty hens in their first week of egg p r o d u c tion were used with 10 hens r a n d o m l y assigned per experimental t r e a t m e n t . T h e hens were inseminated 3X t h e first week of egg p r o d u c tion t h e n weekly for 15 consecutive weeks. Each hen was inseminated with a dose of .1 cc diluted semen containing a p p r o x i m a t e l y 2 7 5 ± 25 million sperm. Sperm c o n c e n t r a t i o n was d e t e r m i n e d for each sample with a KlettS u m m e r s o n p h o t o m e t e r at 4 0 0 t o 4 6 5 jum (Cecil, 1 9 8 1 ) . Eggs were collected daily, s t o r e d at 15 C, a n d i n c u b a t e d within 7 days. Fertility ( F ) was estimated o n a p p r o x i m a t e l y 700 eggs per treatm e n t by candling on Day 7 of i n c u b a t i o n . Fertility was expressed as t h e percentage of viable e m b r y o s . Clear eggs were n o t b r o k e n o p e n and e x a m i n e d macroscopically. Hatchability ( H F ) was d e t e r m i n e d o n Day 2 9 of i n c u b a t i o n and was expressed as t h e p e r c e n t of h a t c h of fertile eggs (as d e t e r m i n e d b y candling o n Day 7). Experiment
4. T w o i n d e p e n d e n t trials were
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a ' ,c Means followed by a different letter are different (P<.05).
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SEXTON AND GIESEN
Experiment 5. This experiment was designed to determine if pH per se or the presence of the HCl used to adjust extender pH was responsible for the results obtained in Experiment 1. The BPSE was prepared and divided into three equal portions. Adjustments of pH from 7.5 to 6.5 was done with either concentrated HCl, phosphoric acid (85%), or glacial acetic acid. After the pH was adjusted to 6.5 ± .03, osmolarity for each portion was adjusted to 352 ± 1 with H20. Undiluted semen (3 cc) was divided into 3 equal parts and diluted with the appropriate extender. Each diluted sample was further divided into two equal portions with one portion used immediately to inseminate the control hens (0 hr) and the other portion was held at 15 C for 6 hr. The samples held at 15 C were agitated as described in Experiment 4. Sixty hens in the first week of egg production were used with 10 hens randomly assigned per experimental treatment. The insemination schedule was similar to that outlined for Experiment 3 except the experimental duration period was 10 weeks. Each hen was inseminated with a dose of .1 cc of diluted semen containing approximately 330 ± 20 million sperm. Fertility was estimated on 400 eggs per treatment. Egg handling methods, fertility, and hatchability determinations were similar to those for Experiment 3. Percentage data (motility, fertility, and hatchability) were transformed to arcsin and
significance was determined at the 5% level of probability by analysis of variance (AOV). Differences between treatment means were estimated by the least significance difference test. Standard error of the mean (SEM) were calculated from nontransformed data.
RESULTS AND DISCUSSION
The selection of the two pH (7.5 and 6.5) and osmolarity (355 and 280 mOsml) extender limits was based on published work. Several researchers have reported small variations in fertility with chicken or turkey semen stored in extenders at pH 6.5 to 7.5 and osmotic pressures of 250 to 450 mOsm (e.g., Hobbs and Harris, 1963; Harris and Hobbs, 1968; Graham and Brown, 1971; Van Wambeke, 1977; Lake andRavie, 1979). Previous workers have reported that pH does influence the respiration rate of chicken sperm; maximum respiratory activity was observed at pH 7.0 (Lardy and Phillips, 1943; Bogdonoff and Shaffner, 1954). Ogasawara and Lorenz (1964) showed that pH 6.0 reduced metabolic activity of chicken sperm, as measured by direct oxygen uptake, but metabolic activity was less affected at pH 8.0. However, the data in the present study (Table 1) indicated that the accumulative respiration of turkey sperm, at 15 C for 6 hr, diluted in BPSE pH 6.5 was 13% lower (P<.05) than sperm diluted in the 7.5 pH extender, but only at the low osmotic pressure of 280 mOsm. No relationship was noted in sperm respiration and extender pH at the high (355 mOsm) osmotic pressure. These data are the first reported evidence of the effect of osmotic pressure on turkey sperm respiration. The data in Table 2 clearly indicate that extender pH influences sperm motility while extender osmolarity does not. Sperm diluted in BPSE pH 6.5, regardless of osmolarity, were significantly less motile than those diluted in BPSE at pH 7.5, regardless of holding time (0 or 6 hr). Lake and Ravie (1979) reported that chicken sperm retained maximum motility at pH 7.4, and only moderate motility was seen when sperm were stored at pH 6.8. A similar low pH effect on chicken sperm motility had been observed by Bogdonoff and Shaffner (1954) and Hobbs and Harris (1963). With the exception of sperm diluted with pH 6.5 — 280 mOsm extender, holding at 15 C for 6 hr did not lower motility.
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designed to determine the influence of semen sample agitation during holding at 15 C. In one trial, 2 cc of undiluted semen was divided into 2 equal parts and diluted 1:1 with BPSE (pH 7.5 — osmolarity 355). In the other trial the semen sample was diluted 1:1 with BPSE (pH 6.5 — osmolarity 355). In both trials one part was agitated on a Lab-Line Orbit Shaker at 150 rpm, while the other sample served as the nonagitated control. Sixty hens in their 7th week of egg production were used in both trials with 10 hens randomly assigned per treatment. The insemination schedule was similar to that outlined for Experiment 3 except the experimental duration was 10 weeks. Each hen was inseminated with a dose of .1 cc of diluted semen containing approximately 288 ± 1 5 million sperm. Fertility was estimated on 375 eggs per treatment. Egg handling methods, fertility, and hatchability determinations were similar to those outlined for Experiment 3.
BELTSVILLE POULTRY SEMEN EXTENDER
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TABLE 2. Influence of extender pH and osmolality on progressive motility of turkey semen held at 15 C for 0 (—) or 6 (+) hr (.Experiment 2) Modified BPSE
Progressive motility (%)'
pH
mOsm
7.5 7.5 6.5 6.5
355 280 280 355
SEM
(-)
(+)
73 ± 3 C 76 ± 2C 56 ± 5 b 57±5b
70 68 44 52
± 3C ± 4C + 5ab ± 5b
6
1
Mean and SEM of 15 determinations.
The effect of pH on sperm fertility was quite the opposite of the effect of pH on sperm motility. The fertilizing capacity of sperm diluted and stored in either of the pH 6.5 extenders was not significantly different than the unstored controls (Table 3), whereas the fertility of semen stored for 6 hr in either of the pH 7.5 extenders was lower than the unstored controls. Apparently the relationship of extender pH to the maintenance of fertility of turkey sperm during low temperature storage is similar to that for chicken sperm. Lake and Ravie (1979) reported satisfactory fertility (87%) when chicken sperm was stored in either pH 6.8 or 7.1 extender for 24 hr at 5 C. Changes in osmotic pressure of the extender had little influence on the fertilizing capacity of either unstored or stored sperm. The data in Table 1, Table 2, and Table 3 clearly demonstrate that there is not always a simple relationship between the fertilizing ability of sperm ind other functions of the cell such as motility and/or respiratory activity. Lake and Ravie (1979) concluded that the direct effect of pH on spermatozoa may be to regulate the activity of the sperm cell. In the present study, pH 6.5 extender appeared to regulate, by decreasing, the motility of the sperm cell during storage in vitro while having little influence on other cellular activities such as respiration and fertilizing capacity. Conversely, pH 7.5 extenders lowered fertilizing capacity while having no influence on respiration or motility. The fertility results presented in Table 3 reveal an interesting phenomenon. The fertilizing capacity of semen diluted with the pH 7.5
extenders and stored at 15 C was very high (both 92%) the first 5 weeks of egg production. However, as the laying season progressed, the fertility of these samples decreased below that of the unstored control, 63 and 74% during weeks 11 to 15. Harper (1955) reported similar results with undiluted semen held 0, 1, 2, or 4 hr before AI. The present results are difficult to explain, particularly since such a phenomenon was not observed with semen stored in the pH 6.5 extenders. Nonetheless, the results of this study and those of Harper (1955) emphasize the importance of conducting long-term AI trials with studies involving turkey semen preservation to obtain an accurate assessment of the experimental treatments. While agitation of bull semen samples during storage at low temperatures has received some research attention (Prince and Almquist, 1948; Foote, 1967), information on the effects of agitating avian sperm during storage was nonexistent. The data reported in Table 4 clearly show that constant agitation during storage had a beneficial effect when semen was diluted in the pH 7.5 extender but had no effect when semen was diluted in the pH 6.5 extender. The results of this study make it difficult to explain how agitation influences sperm viability. However, several plausible explanations can be given. Lake (1978) observed that the pH of diluted turkey semen became progressively more alkaline (as high as pH 7.6), which led to a decline in fertilizing ability. Agitation of semen samples diluted in pH 7.5 BPSE may have prevented the accumulation of metabolic by-products which could impair sperm viabil-
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Means within rows followed by a different superscript are different (P<.05) than the unstored control. b ' c Means within columns followed by a different superscript are different (P<.05).
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SEXTON AND GIESEN
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The effect of semen storage treatments on hatch of fertile eggs (HF) was inconsistent from experiment to experiment. In Experiment 3, HF for hens inseminated with semen diluted in BPSE (pH 6.5, 355 mOsm) and stored 6 hr was significantly higher than the unstored control (Table 3). However, in other experiments (Experiments 4 and 5) conducted under identical conditions the results indicated that semen treatments had little influence on HF (Tables
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ity. Also, the beneficial effect of agitation may have been the result of increased aeration of the semen sample. Several researchers (Proudfoot, 1966; Proudfoot and Stewart, 1967; Van Wambeke, 1968) demonstrated the importance of aerobic conditions for maintaining the fertilizing capacity of stored chicken semen; turkey semen may have similar requirements. As stated earlier, concentrated HC1 was used to lower the pH from 7.5 to 6.5. It was not possible to establish whether the results reported in Table 3 for the pH 6.5 extenders was due to pH per se or the presence of the additional chloride ions. The results presented in Table 5 are also somewhat inconclusive. Phosphoric acid, when used to adjust pH, had no effect on fertility, whereas acetic and hydrochloric acid lowered the fertilizing capacity of stored semen. The latter result was surprising since in the previous two experiments (Tables 3 and 4) fertility of semen was not affected when stored in BPSE which had been adjusted to pH 6.5 with concentrated HC1. Perhaps the strain of birds used in the experiments had some influencing effect, because Small Whites were used in Experiments 3 and 4 whereas Large Whites were used in Experiment 5. The depressive affect of HC1 or acetic acid on fertility cannot be explained by their effect on sperm motility. For example, acetic acid severely depressed the motility of unstored sperm but had no effect on fertility. Lack of a correlation between turkey sperm motility and fertility has been observed previously (Cherms, 1968). Acetate has been shown to increase the metabolic activity of chicken sperm (Scott et al, 1962). Several preliminary studies have indicated that acetic acid adjusted BPSE had no influence on respiration of turkey sperm but causes the sperm cell to decrease in size (unpublished observations). Damage to the integrity of the sperm cell by acetic acid could account for the low fertility reported in Experiment 5.
VsU
h
BELTSVILLE POULTRY SEMEN EXTENDER
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TABLE 4. Effect of sample agitation on reproductive parameters of turkey semen diluted in two extenders and held at 15 C for 0 and 6 hr (Experiment 4)1 Percent Sample agitation
BPSE (6.5 - 355)
BPSE (7.5 - 355)
Storage (hr)
No No Yes
F2
HF 2
F
HF
96* 87b 92a
89 91 90
96 90 95
93 92 95
SEM ' Means within columns followed by a different superscript are different (P<.05).
1
Composite of two independent trials. Each value is the mean of 10 weekly inseminations.
2
F = candling fertility; HF = hatch of fertile eggs.
TABLE 5. Influence of various acids used to adjust extender pH on reproductive parameters of turkey semen held at 15 C for 0 (—) or 6 (+) hr (Experiment 5) Percent 1 1Progressive motility
Hatch of fertiles
Candling fertility
BPSE2 adjusted with
(-)
(+)
(-)
(+)
(-)
(+)
Hydrochloric acid Phosphoric acid Acetic acid
59b 42<= 27d
63b 52bd 48ac
94 91 95
87 a 89 76ab
85 84 87
84 86 88
SEM a
6
2
2
Means within rows within parameter having superscript a are different from 0 (—) hr (P<.05). ' ' Means within columns within parameters followed by a different superscript are different (P<.05).
1
Mean and SEM of 10 determinations.
2
pH = 6.5 and osmolarity = 352.
4 and 5). F u r t h e r e x p e r i m e n t s are n e e d e d t o clarify these discrepancies. In conclusion, t h e results of these studies have s h o w n t h a t t u r k e y semen can be stored u p t o 6 h in vitro w i t h o u t a loss of fertilizing capacity w h e n diluted in p H 6.5 BPSE. Such a semen preservation program if developed and utilized b y commercial t u r k e y breeders could significantly r e d u c e t h e cost of AI.
ACKNOWLEDGMENTS T h e a u t h o r s wish t o express appreciation t o
D. M. Gavelek a n d V. W. S m o o t for their technical assistance.
REFERENCES Bogdonoff, P. D., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic activity and fertilizing capacity of chicken semen. Poultry Sci. 33:665-669. Cecil, H., 1981. Effects of dietary protein on body weight and reproductive performance of male turkeys. Poultry Sci. 60:1049-1055. Cherms, F. L., 1968. Variations in semen quality and the relationship of semen quality to fertility in turkeys. Poultry Sci. 47:746-754.
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a
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SEXTON AND GIESEN Ogasawara, F. X., and F. W. Lorenz, 1964. Respiratory rate of cock spermatozoa as affected by oviduct extracts. J. Reprod. Fertil. 7:281-288. Prince, P. W., and J. O. Almquist, 1948. The effect of agitation upon the livability of bovine spermatozoa. J. Dairy Sci. 31:839-844. Proudfoot, F. G., 1966. The influence of oxygen and other gases on the fertilizing ability of fowl semen held several hours at 10°C in cryovac enclosures. Poultry Sci. 45:443—446. Proudfoot, F. G., and D.K.R. Stewart, 1967. The effect of oxygen and type of container on the retention of the fertilizing capacity of fowl spermatozoa stored in vitro. J. Reprod. Fertil. 13:251-257. Scott, T. W., I. G. White, and E. F. Annison, 1962. Glucose and acetate metabolism by ram, bull, dog and fowl spermatozoa. Biochem. J. 83:398—404. Sexton, T. J., 1980. A new poultry semen extender. 5. Relationship of diluent components to cytotoxic effects of dimethylsulfoxide on turkey spermatozoa. Poultry Sci. 59:1142—1144. Van Wambeke, F., 1968. The effect of an oxygenated storage medium on die fertilizing capacity of fowl spermatozoa stored for different periods. 8th Int. Congr. Anim. Reprod. Artif. Insem., Paris 2:1645-1647. Van Wambeke, F., 1977. The effect of tonicity of storage media for fowl semen on the occurrence of neck-bending spermatozoa, fertility and hatchability. Br. Poultry Sci. 18:163-168. Wilcox, F. H., and C. S. Shaffner, 1960. Storage of turkey semen. Poultry Sci. 39:1580-1581.
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Foote, R. H., 1967. Influence of light and agitation on bovine spermatozoa stored with protective agents. J. Dairy Sci. 50:1468-1474. Graham, E. F., and K. I. Brown, 1971. Effect of osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poultry Sci. 50:836-838. Harper, J. A., 1955. The effect of holding time of turkey semen on fertilizing capacity. Poultry Sci. 34:1289-1291. Harris, G. C., and T. D. Hobbs, 1968. Effects of freezing point depression and fluid to gas ratio on fertility of fowl spermatozoa stored in COa extenders. J. Reprod. Fertil. 16:389-394. Hobbs, T. D., and G. C. Harris, 1963. Effect of freezing point depression and pH on motility and fertility of chicken spermatozoa. Poultry Sci. 42: 254-259. Johnston, N. P., 1977. Midnight insemination increases fertility. Turkey World 52:28-29. Lake, P. E., 1978. The dilution and storage of turkey semen. Turkeys Nov./Dec. Suppl. Lake, P. E., and O. Ravie, 1979. Effect on fertility of storing fowl semen for 24 hr at 5°C in fluids of different pH. J. Reprod. Fertil. 57:149-155. Lardy, H. A., and P. H. Phillips, 1943. Effect of pH and certain electrolytes on the metabolism of ejaculated spermatozoa. Am. J. Physiol. 138: 741. Litjen, J. B., 1972. Artificial insemination in turkeys and an investigation into a suitable diluent for turkey semen. 8th Int. Congr. Anim. Reprod. Art. Insem., Munich 3:1698.
1982 Poultry Science 61:1202-1208
INTRODUCTION Artificial insemination (AI) costs t h e US t u r k e y breeding industry a p p r o x i m a t e l y 12 million dollars annually. Semen e x t e n d e r s have been used t o lower the cost of AI b y allowing diluted semen t o be inseminated into m o r e hens t h a n is possible with u n d i l u t e d semen. T h e savings in p r o d u c t i o n costs o b t a i n e d by using e x t e n d e r s is realized because fewer t o m s are required. Additional cost savings could c o m e from short-term holding (6 t o 8 hr) of semen prior t o AI with reduced labor costs and increased p r o d u c t i o n efficiency. Advantages would include t h e t r a n s p o r t of semen over short distances, the establishment of breeder torn farms, b e t t e r utilization of t h e AI labor force, a n d t h e insemination of h e n s late in t h e a f t e r n o o n , a time f o u n d to be m o s t desirable (Johnston, 1977).
1 Scientific Article No. 12-81-10083 of the Alabama Agricultural Experiment Station.
F e w investigations have been c o n d u c t e d on the dilution and storage of t u r k e y semen. Results of past w o r k e r s have indicated t h a t the fertilizing capacity of diluted t u r k e y semen decreased rapidly after 1 or 2 hr storage in vitro (Wilcox and Shaffner, I 9 6 0 ; G r a h a m and Brown, 1 9 7 1 ; Litjen, 1 9 7 2 ) . However, a recent r e p o r t b y Lake ( 1 9 7 8 ) indicated t h a t satisfact o r y fertility levels of 85 t o 8 8 % can be obtained from a single insemination of diluted t u r k e y semen held 5 h r before AI. Preliminary data^ from this l a b o r a t o r y indicated t h a t reasonably good fertility (73%) can be obtained from weekly inseminations with turkey semen diluted in Beltsville P o u l t r y Semen E x t e n d e r (BPSE) and held 6 hr before AI (Sexton, unpublished d a t a ) . T h e primary p u r p o s e of this e x p e r i m e n t was t o measure t h e fertilizing capacity of t u r k e y semen diluted in t h e original BPSE and in three modifications (pH and osmolarity) of BPSE. T h e semen was held for 0 or 6 hr before AI. A secondary objective was t o d e t e r m i n e t h e influence of agitating diluted semen, during holding at 15 C.
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ABSTRACT A series of experiments were conducted to measure the effects of various combinations of extender pH (6.5 vs. 7.5), osmolarity (280 us. 355 mOsmols), and sample agitation on the fertilizing capacity of turkey semen held for 6 hr at 15 C. Respiration of turkey sperm diluted in Beltsville Poultry Semen Extender (BPSE) to pH 6.0 and 280 mOsmols was lower than that of sperm diluted in BPSE with pH 7.5 and 280 mOsmols (mOsm). Sperm respiration was unaffected by extender pH adjusted to 350 mOsm, regardless of pH. Motility of both unstored and stored sperm was significantly lower in BPSE at pH 6.5 than at pH 7.5. The fertilizing capacity of sperm diluted and stored in pH 6.5 extenders did not differ from the fertilizing capacity of the unstored controls. The fertility of unstored sperm diluted in the pH 7.5 extenders did not differ from the controls, but holding for 6 hr at 15 C resulted in lower fertility. Extender osmolarity in the range studied had no influence on sperm motility, respiration, or fertility. Phosphoric acid, when used to adjust pH from 7.5 to 6.5, had no effect on fertility, whereas acetic and hydrochloric acid lowered the fertilizing capacity of stored semen. Sample agitation was critical for maintenance of sperm fertility in samples diluted and stored in pH 7.5 extenders but had no effect on sperm diluted in pH 6.5 extenders. (Key words: diluent, turkey, pH, osmolarity, storage, semen)
BELTSVILLE POULTRY SEMEN EXTENDER TABLE 1. Effect of extender pH and osmolality on oxygen uptake (02) of turkey semen incubated at 15 C for 6 hr (Experiment 1)
pH
mOsm
Accumulative 0 2 uptake/10 9 spermatozoa 1
7.5 7.5 6.5 6.5
355 280 280 355
26.1ac 29.9 a 23.7bc 26.5 a c
Modified BPSE
SEM
2.0
1
Mean and SEM of 6 determinations.
MATERIALS AND METHODS In E x p e r i m e n t s 1 t h r o u g h 4 , second season, nonforce-molted males (Large White) and first season females (Small White) of commercial strains were used, while in E x p e r i m e n t 5 first season males (Large White) a n d first season females (Large White) were used. All five experim e n t s were c o n d u c t e d sequentially. Semen samples were collected from a t least 10 males, pooled immediately after collection, and diluted 1 p a r t semen t o 1 part of e x t e n d e r . All semen samples were diluted within 10 min of semen collection. T h e c o m p o s i t i o n of BPSE for t u r k e y s has been outlined by Sexton ( 1 9 8 0 ) . T h e p H and osmolarity changes were t h e only modifications m a d e t o t h e original BPSE, which has a p H of 7.5 and osmolarity of 355 (mOsm/1 H 2 0 ) . T h e BPSE p H was adjusted from 7.5 t o 6.5 in t w o modified versions by the addition of concentrated HC1 (3 to 4 ml/liter of BPSE), while osmolarity was adjusted t o either 355 or 2 8 0 m O s m b y t h e addition of 9 0 0 or 1300 ml of water, respectively. Regardless of the a m o u n t of water used t o adjust osmolarity, t h e ratio of c o m p o n e n t s in each of t h e modified versions was t h e same. D e t e r m i n a t i o n s of p H a n d o s m o larity were m a d e with a Corning Model 110 p H m e t e r and an O s m e t t e A a u t o m a t i c o s m o m e t e r . T h e pH-osmolarity combinations tested are listed in Table 1. Experiments 1 to 3. T h r e e i n d e p e n d e n t e x p e r i m e n t s were designed t o assess t h e effects of dilution of t u r k e y semen in regular and modified versions of BPSE and held at 15 C for 6 h. In E x p e r i m e n t 1, sperm respiration, sperm
motility, egg fertility, and hatchability were measured. Cumulative sperm respiration (jA 0 2 u p t a k e / 1 0 9 sperm) of diluted t u r k e y sperm over a 6 hr period was d e t e r m i n e d m a n o metrically, hourly for 6 hr, with a Gilson Refrigerated Differential Respirometer, Model GR14. Diluted semen (.5 cc containing a p p r o x i m a t e l y 1.5 X 1 0 9 sperm) was placed in t h e main chamber of a 7 ml capacity i n c u b a t i o n flask within 30 min of semen collection. T h e center well of each flask contained .2 cc K O H (20% w/v) t o absorb t h e C 0 2 p r o d u c e d during incubation. Flasks were equilibrated at 15 C for 10 m i n , t h e n i n c u b a t e d with c o n s t a n t agitation at 8 0 cycle/min. E x p e r i m e n t 2 was designed t o d e t e r m i n e t h e effect of e x t e n d e r pH-osmolarity o n t h e motility of t u r k e y sperm held 6 hr at 15 C. T w o cubic centimeters of semen diluted in one of four e x t e n d e r s were placed in a 10 cc flask, covered lightly with a l u m i n u m foil, and held at 15 C for 6 hr. At 0 and 6 hr, flask c o n t e n t s were mixed, an 5 All aliquot was removed, and progressive motility was estimated on a scale of 0 to 100, microscopically (phase-contrast*) at 4 3 0 x on a microscope slide m a i n t a i n e d at r o o m temperature. In E x p e r i m e n t 3, 4 cc of u n d i l u t e d semen was divided into 4 equal parts, diluted with t h e a p p r o p r i a t e e x t e n d e r , and held at 15 C as described for E x p e r i m e n t 2. One p a r t was used immediately t o inseminate t h e c o n t r o l hens (0 hr) and t h e o t h e r part was held for 6 hr. Eighty hens in their first week of egg p r o d u c tion were used with 10 hens r a n d o m l y assigned per experimental t r e a t m e n t . T h e hens were inseminated 3X t h e first week of egg p r o d u c tion t h e n weekly for 15 consecutive weeks. Each hen was inseminated with a dose of .1 cc diluted semen containing a p p r o x i m a t e l y 2 7 5 ± 25 million sperm. Sperm c o n c e n t r a t i o n was d e t e r m i n e d for each sample with a KlettS u m m e r s o n p h o t o m e t e r at 4 0 0 t o 4 6 5 jum (Cecil, 1 9 8 1 ) . Eggs were collected daily, s t o r e d at 15 C, a n d i n c u b a t e d within 7 days. Fertility ( F ) was estimated o n a p p r o x i m a t e l y 700 eggs per treatm e n t by candling on Day 7 of i n c u b a t i o n . Fertility was expressed as t h e percentage of viable e m b r y o s . Clear eggs were n o t b r o k e n o p e n and e x a m i n e d macroscopically. Hatchability ( H F ) was d e t e r m i n e d o n Day 2 9 of i n c u b a t i o n and was expressed as t h e p e r c e n t of h a t c h of fertile eggs (as d e t e r m i n e d b y candling o n Day 7). Experiment
4. T w o i n d e p e n d e n t trials were
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a ' ,c Means followed by a different letter are different (P<.05).
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SEXTON AND GIESEN
Experiment 5. This experiment was designed to determine if pH per se or the presence of the HCl used to adjust extender pH was responsible for the results obtained in Experiment 1. The BPSE was prepared and divided into three equal portions. Adjustments of pH from 7.5 to 6.5 was done with either concentrated HCl, phosphoric acid (85%), or glacial acetic acid. After the pH was adjusted to 6.5 ± .03, osmolarity for each portion was adjusted to 352 ± 1 with H20. Undiluted semen (3 cc) was divided into 3 equal parts and diluted with the appropriate extender. Each diluted sample was further divided into two equal portions with one portion used immediately to inseminate the control hens (0 hr) and the other portion was held at 15 C for 6 hr. The samples held at 15 C were agitated as described in Experiment 4. Sixty hens in the first week of egg production were used with 10 hens randomly assigned per experimental treatment. The insemination schedule was similar to that outlined for Experiment 3 except the experimental duration period was 10 weeks. Each hen was inseminated with a dose of .1 cc of diluted semen containing approximately 330 ± 20 million sperm. Fertility was estimated on 400 eggs per treatment. Egg handling methods, fertility, and hatchability determinations were similar to those for Experiment 3. Percentage data (motility, fertility, and hatchability) were transformed to arcsin and
significance was determined at the 5% level of probability by analysis of variance (AOV). Differences between treatment means were estimated by the least significance difference test. Standard error of the mean (SEM) were calculated from nontransformed data.
RESULTS AND DISCUSSION
The selection of the two pH (7.5 and 6.5) and osmolarity (355 and 280 mOsml) extender limits was based on published work. Several researchers have reported small variations in fertility with chicken or turkey semen stored in extenders at pH 6.5 to 7.5 and osmotic pressures of 250 to 450 mOsm (e.g., Hobbs and Harris, 1963; Harris and Hobbs, 1968; Graham and Brown, 1971; Van Wambeke, 1977; Lake andRavie, 1979). Previous workers have reported that pH does influence the respiration rate of chicken sperm; maximum respiratory activity was observed at pH 7.0 (Lardy and Phillips, 1943; Bogdonoff and Shaffner, 1954). Ogasawara and Lorenz (1964) showed that pH 6.0 reduced metabolic activity of chicken sperm, as measured by direct oxygen uptake, but metabolic activity was less affected at pH 8.0. However, the data in the present study (Table 1) indicated that the accumulative respiration of turkey sperm, at 15 C for 6 hr, diluted in BPSE pH 6.5 was 13% lower (P<.05) than sperm diluted in the 7.5 pH extender, but only at the low osmotic pressure of 280 mOsm. No relationship was noted in sperm respiration and extender pH at the high (355 mOsm) osmotic pressure. These data are the first reported evidence of the effect of osmotic pressure on turkey sperm respiration. The data in Table 2 clearly indicate that extender pH influences sperm motility while extender osmolarity does not. Sperm diluted in BPSE pH 6.5, regardless of osmolarity, were significantly less motile than those diluted in BPSE at pH 7.5, regardless of holding time (0 or 6 hr). Lake and Ravie (1979) reported that chicken sperm retained maximum motility at pH 7.4, and only moderate motility was seen when sperm were stored at pH 6.8. A similar low pH effect on chicken sperm motility had been observed by Bogdonoff and Shaffner (1954) and Hobbs and Harris (1963). With the exception of sperm diluted with pH 6.5 — 280 mOsm extender, holding at 15 C for 6 hr did not lower motility.
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designed to determine the influence of semen sample agitation during holding at 15 C. In one trial, 2 cc of undiluted semen was divided into 2 equal parts and diluted 1:1 with BPSE (pH 7.5 — osmolarity 355). In the other trial the semen sample was diluted 1:1 with BPSE (pH 6.5 — osmolarity 355). In both trials one part was agitated on a Lab-Line Orbit Shaker at 150 rpm, while the other sample served as the nonagitated control. Sixty hens in their 7th week of egg production were used in both trials with 10 hens randomly assigned per treatment. The insemination schedule was similar to that outlined for Experiment 3 except the experimental duration was 10 weeks. Each hen was inseminated with a dose of .1 cc of diluted semen containing approximately 288 ± 1 5 million sperm. Fertility was estimated on 375 eggs per treatment. Egg handling methods, fertility, and hatchability determinations were similar to those outlined for Experiment 3.
BELTSVILLE POULTRY SEMEN EXTENDER
1205
TABLE 2. Influence of extender pH and osmolality on progressive motility of turkey semen held at 15 C for 0 (—) or 6 (+) hr (.Experiment 2) Modified BPSE
Progressive motility (%)'
pH
mOsm
7.5 7.5 6.5 6.5
355 280 280 355
SEM
(-)
(+)
73 ± 3 C 76 ± 2C 56 ± 5 b 57±5b
70 68 44 52
± 3C ± 4C + 5ab ± 5b
6
1
Mean and SEM of 15 determinations.
The effect of pH on sperm fertility was quite the opposite of the effect of pH on sperm motility. The fertilizing capacity of sperm diluted and stored in either of the pH 6.5 extenders was not significantly different than the unstored controls (Table 3), whereas the fertility of semen stored for 6 hr in either of the pH 7.5 extenders was lower than the unstored controls. Apparently the relationship of extender pH to the maintenance of fertility of turkey sperm during low temperature storage is similar to that for chicken sperm. Lake and Ravie (1979) reported satisfactory fertility (87%) when chicken sperm was stored in either pH 6.8 or 7.1 extender for 24 hr at 5 C. Changes in osmotic pressure of the extender had little influence on the fertilizing capacity of either unstored or stored sperm. The data in Table 1, Table 2, and Table 3 clearly demonstrate that there is not always a simple relationship between the fertilizing ability of sperm ind other functions of the cell such as motility and/or respiratory activity. Lake and Ravie (1979) concluded that the direct effect of pH on spermatozoa may be to regulate the activity of the sperm cell. In the present study, pH 6.5 extender appeared to regulate, by decreasing, the motility of the sperm cell during storage in vitro while having little influence on other cellular activities such as respiration and fertilizing capacity. Conversely, pH 7.5 extenders lowered fertilizing capacity while having no influence on respiration or motility. The fertility results presented in Table 3 reveal an interesting phenomenon. The fertilizing capacity of semen diluted with the pH 7.5
extenders and stored at 15 C was very high (both 92%) the first 5 weeks of egg production. However, as the laying season progressed, the fertility of these samples decreased below that of the unstored control, 63 and 74% during weeks 11 to 15. Harper (1955) reported similar results with undiluted semen held 0, 1, 2, or 4 hr before AI. The present results are difficult to explain, particularly since such a phenomenon was not observed with semen stored in the pH 6.5 extenders. Nonetheless, the results of this study and those of Harper (1955) emphasize the importance of conducting long-term AI trials with studies involving turkey semen preservation to obtain an accurate assessment of the experimental treatments. While agitation of bull semen samples during storage at low temperatures has received some research attention (Prince and Almquist, 1948; Foote, 1967), information on the effects of agitating avian sperm during storage was nonexistent. The data reported in Table 4 clearly show that constant agitation during storage had a beneficial effect when semen was diluted in the pH 7.5 extender but had no effect when semen was diluted in the pH 6.5 extender. The results of this study make it difficult to explain how agitation influences sperm viability. However, several plausible explanations can be given. Lake (1978) observed that the pH of diluted turkey semen became progressively more alkaline (as high as pH 7.6), which led to a decline in fertilizing ability. Agitation of semen samples diluted in pH 7.5 BPSE may have prevented the accumulation of metabolic by-products which could impair sperm viabil-
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Means within rows followed by a different superscript are different (P<.05) than the unstored control. b ' c Means within columns followed by a different superscript are different (P<.05).
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SEXTON AND GIESEN
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The effect of semen storage treatments on hatch of fertile eggs (HF) was inconsistent from experiment to experiment. In Experiment 3, HF for hens inseminated with semen diluted in BPSE (pH 6.5, 355 mOsm) and stored 6 hr was significantly higher than the unstored control (Table 3). However, in other experiments (Experiments 4 and 5) conducted under identical conditions the results indicated that semen treatments had little influence on HF (Tables
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ity. Also, the beneficial effect of agitation may have been the result of increased aeration of the semen sample. Several researchers (Proudfoot, 1966; Proudfoot and Stewart, 1967; Van Wambeke, 1968) demonstrated the importance of aerobic conditions for maintaining the fertilizing capacity of stored chicken semen; turkey semen may have similar requirements. As stated earlier, concentrated HC1 was used to lower the pH from 7.5 to 6.5. It was not possible to establish whether the results reported in Table 3 for the pH 6.5 extenders was due to pH per se or the presence of the additional chloride ions. The results presented in Table 5 are also somewhat inconclusive. Phosphoric acid, when used to adjust pH, had no effect on fertility, whereas acetic and hydrochloric acid lowered the fertilizing capacity of stored semen. The latter result was surprising since in the previous two experiments (Tables 3 and 4) fertility of semen was not affected when stored in BPSE which had been adjusted to pH 6.5 with concentrated HC1. Perhaps the strain of birds used in the experiments had some influencing effect, because Small Whites were used in Experiments 3 and 4 whereas Large Whites were used in Experiment 5. The depressive affect of HC1 or acetic acid on fertility cannot be explained by their effect on sperm motility. For example, acetic acid severely depressed the motility of unstored sperm but had no effect on fertility. Lack of a correlation between turkey sperm motility and fertility has been observed previously (Cherms, 1968). Acetate has been shown to increase the metabolic activity of chicken sperm (Scott et al, 1962). Several preliminary studies have indicated that acetic acid adjusted BPSE had no influence on respiration of turkey sperm but causes the sperm cell to decrease in size (unpublished observations). Damage to the integrity of the sperm cell by acetic acid could account for the low fertility reported in Experiment 5.
VsU
h
BELTSVILLE POULTRY SEMEN EXTENDER
1207
TABLE 4. Effect of sample agitation on reproductive parameters of turkey semen diluted in two extenders and held at 15 C for 0 and 6 hr (Experiment 4)1 Percent Sample agitation
BPSE (6.5 - 355)
BPSE (7.5 - 355)
Storage (hr)
No No Yes
F2
HF 2
F
HF
96* 87b 92a
89 91 90
96 90 95
93 92 95
SEM ' Means within columns followed by a different superscript are different (P<.05).
1
Composite of two independent trials. Each value is the mean of 10 weekly inseminations.
2
F = candling fertility; HF = hatch of fertile eggs.
TABLE 5. Influence of various acids used to adjust extender pH on reproductive parameters of turkey semen held at 15 C for 0 (—) or 6 (+) hr (Experiment 5) Percent 1 1Progressive motility
Hatch of fertiles
Candling fertility
BPSE2 adjusted with
(-)
(+)
(-)
(+)
(-)
(+)
Hydrochloric acid Phosphoric acid Acetic acid
59b 42<= 27d
63b 52bd 48ac
94 91 95
87 a 89 76ab
85 84 87
84 86 88
SEM a
6
2
2
Means within rows within parameter having superscript a are different from 0 (—) hr (P<.05). ' ' Means within columns within parameters followed by a different superscript are different (P<.05).
1
Mean and SEM of 10 determinations.
2
pH = 6.5 and osmolarity = 352.
4 and 5). F u r t h e r e x p e r i m e n t s are n e e d e d t o clarify these discrepancies. In conclusion, t h e results of these studies have s h o w n t h a t t u r k e y semen can be stored u p t o 6 h in vitro w i t h o u t a loss of fertilizing capacity w h e n diluted in p H 6.5 BPSE. Such a semen preservation program if developed and utilized b y commercial t u r k e y breeders could significantly r e d u c e t h e cost of AI.
ACKNOWLEDGMENTS T h e a u t h o r s wish t o express appreciation t o
D. M. Gavelek a n d V. W. S m o o t for their technical assistance.
REFERENCES Bogdonoff, P. D., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic activity and fertilizing capacity of chicken semen. Poultry Sci. 33:665-669. Cecil, H., 1981. Effects of dietary protein on body weight and reproductive performance of male turkeys. Poultry Sci. 60:1049-1055. Cherms, F. L., 1968. Variations in semen quality and the relationship of semen quality to fertility in turkeys. Poultry Sci. 47:746-754.
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a
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SEXTON AND GIESEN Ogasawara, F. X., and F. W. Lorenz, 1964. Respiratory rate of cock spermatozoa as affected by oviduct extracts. J. Reprod. Fertil. 7:281-288. Prince, P. W., and J. O. Almquist, 1948. The effect of agitation upon the livability of bovine spermatozoa. J. Dairy Sci. 31:839-844. Proudfoot, F. G., 1966. The influence of oxygen and other gases on the fertilizing ability of fowl semen held several hours at 10°C in cryovac enclosures. Poultry Sci. 45:443—446. Proudfoot, F. G., and D.K.R. Stewart, 1967. The effect of oxygen and type of container on the retention of the fertilizing capacity of fowl spermatozoa stored in vitro. J. Reprod. Fertil. 13:251-257. Scott, T. W., I. G. White, and E. F. Annison, 1962. Glucose and acetate metabolism by ram, bull, dog and fowl spermatozoa. Biochem. J. 83:398—404. Sexton, T. J., 1980. A new poultry semen extender. 5. Relationship of diluent components to cytotoxic effects of dimethylsulfoxide on turkey spermatozoa. Poultry Sci. 59:1142—1144. Van Wambeke, F., 1968. The effect of an oxygenated storage medium on die fertilizing capacity of fowl spermatozoa stored for different periods. 8th Int. Congr. Anim. Reprod. Artif. Insem., Paris 2:1645-1647. Van Wambeke, F., 1977. The effect of tonicity of storage media for fowl semen on the occurrence of neck-bending spermatozoa, fertility and hatchability. Br. Poultry Sci. 18:163-168. Wilcox, F. H., and C. S. Shaffner, 1960. Storage of turkey semen. Poultry Sci. 39:1580-1581.
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Foote, R. H., 1967. Influence of light and agitation on bovine spermatozoa stored with protective agents. J. Dairy Sci. 50:1468-1474. Graham, E. F., and K. I. Brown, 1971. Effect of osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poultry Sci. 50:836-838. Harper, J. A., 1955. The effect of holding time of turkey semen on fertilizing capacity. Poultry Sci. 34:1289-1291. Harris, G. C., and T. D. Hobbs, 1968. Effects of freezing point depression and fluid to gas ratio on fertility of fowl spermatozoa stored in COa extenders. J. Reprod. Fertil. 16:389-394. Hobbs, T. D., and G. C. Harris, 1963. Effect of freezing point depression and pH on motility and fertility of chicken spermatozoa. Poultry Sci. 42: 254-259. Johnston, N. P., 1977. Midnight insemination increases fertility. Turkey World 52:28-29. Lake, P. E., 1978. The dilution and storage of turkey semen. Turkeys Nov./Dec. Suppl. Lake, P. E., and O. Ravie, 1979. Effect on fertility of storing fowl semen for 24 hr at 5°C in fluids of different pH. J. Reprod. Fertil. 57:149-155. Lardy, H. A., and P. H. Phillips, 1943. Effect of pH and certain electrolytes on the metabolism of ejaculated spermatozoa. Am. J. Physiol. 138: 741. Litjen, J. B., 1972. Artificial insemination in turkeys and an investigation into a suitable diluent for turkey semen. 8th Int. Congr. Anim. Reprod. Art. Insem., Munich 3:1698.