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Effect of holding and washing on the quality of goat semen
Small Ruminant Research 66 (2006) 51–57
Effect of holding and washing on the quality of goat semen Rafiqul Islam ∗ , K. Ahmed, B.C. Deka Department of Gynaecology, Obstetrics and A.I., College of Veterinary Sciences, Assam Agricultural University, Khanapara, Guwahati 781022, Assam, India Received 14 October 2003; received in revised form 21 June 2005; accepted 21 June 2005 Available online 12 September 2005
Abstract A total of 18 pooled semen samples collected from 4 healthy crossbred (Beetal × Assam Local) bucks, twice weekly for a period of 12 weeks, were processed via three processing methods, viz. Method I (holding after washing: semen sample was washed twice, extended, held for 0, 1, 3 and 5 h and preserved), Method II (washing after holding: semen sample held for 0, 1, 3 and 5 h, washed twice, extended and preserved) and Method III (holding of undiluted semen: semen sample held for 0, 1, 3 and 5 h, extended and preserved). An isotonic Tris buffer was used to wash the spermatozoa with the aid of centrifugation at 3000 rotations per minute (rpm) for 10 min. A Tris–citric acid–fructose–egg yolk extender was used to extend the semen processed with all three processing methods and preserved at 5 ◦ C up to 72 h. Holding of semen at 24 ◦ C in each processing method caused deterioration of the semen quality during preservation, as the holding time increased from 0 (without holding), 1, 3 and 5 h. The initial sperm motility declined from 85.8 to 71.7% in Method I, from 85.8 to 21.7% in Method II and from 84.2 to 45.0% in Method III, as the holding time increased from 0 to 5 h. The corresponding values were 66.7 and 49.2% for Method I, 66.7 and 8.0% for Method II and 42.5 and 25.8% for Method III, following 72 h of preservation. The percentage of live sperm and intact acrosomes also showed a similar decreasing trend as the holding time increased from 0 to 5 h in all processing methods as revealed in percentage of sperm motility. Washing of spermatozoa without a holding period maintained significantly higher sperm motility (66.7%), live sperm counts (83.0%) and incidence of intact acrosomes (79.3%) following 72 h at preservation, compared to the corresponding values (42.5, 65.2 and 73.3% for sperm motility, live sperm counts and intact acrosomes, respectively) recorded in unwashed semen without a holding period. The values recorded for washed semen without holding were also significantly higher than for semen held for 1, 3 and 5 h in all the three processing methods following 72 h of preservation. This study revealed that the holding of semen caused deterioration of the spermatozoa, whereas, washing without holding had a beneficial effect on the viability of spermatozoa in maintaining its storage ability for up to 72 h of preservation at refrigeration temperature (5 ◦ C). © 2005 Published by Elsevier B.V. Keywords: Goat semen; Holding time; Seminal plasma removal; Preservation quality
∗
Corresponding author. Present address: Division of Animal Reproduction, Gynaecology and Obstetrics, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology (K), Shuhama, Alusteng, Srinagar 190006, Kashmir, India. Tel.: +91 194 2262208/2262209 (o); fax: +91 194 2262207/2262210. E-mail address: [email protected] (R. Islam). 0921-4488/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2005.06.034
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1. Introduction Goat spermatozoa are susceptible to cold shock, which causes the death of numerous spermatozoa during processing for preservation at lower temperature, although the degree of susceptibility is less than that experienced in boar spermatozoa (Sahni and Roy, 1972). Holding, a procedure of retaining or incubation of semen at different room temperatures (18–30 ◦ C) for a few hours before or after processing in boar semen prior to preservation, is generally beneficial (Graham et al., 1971; Pursel et al., 1973; Tamuli, 1993; Nath et al., 1994). Thus, it may be expected that a similar procedure may have some beneficial effect on goat spermatozoa and help sperms to develop a possible resistance against cold shock. However, there is no information currently available on the effect of the holding on the quality of goat semen during preservation at refrigeration temperature (5 ◦ C). An egg yolk-coagulating enzyme is present in the seminal plasma of goat semen, which has limited the use of egg yolk containing extenders for the preservation of goat semen (Roy, 1957). Removal of the seminal plasma by washing the spermatozoa with an isotonic buffer has been reported to improve the quality of goat semen during preservation at various temperatures (Irritani et al., 1961; Deka and Rao, 1986; Misra et al., 1993). However, the effect of the removal of seminal plasma on the quality of goat semen during preservation following holding has not been reported. This study was performed to determine the effect of holding, with or without seminal plasma, on the quality of goat semen for preservation at 5 ◦ C.
2. Materials and methods Four healthy crossbred bucks (Beetal × Assam Local) approximately, 2.5 years of age were selected from the Goat Research Station, Assam Agricultural University, Burnihat, Assam, India. The animals were maintained under standard managemental conditions practiced on the farm. Semen samples were collected twice weekly in the morning from each buck with the aid of the artificial vagina for a period of 12 weeks. Immediately after collection, each ejaculate was evaluated for mass motility (based on the numerical scale 0–4) and initial motility as per the method described by
Zemjanis (1970). Semen samples showing +3 or more mass motility and 80% or more initial motility were pooled. A total of 18 pooled semen samples were used for the study. 2.1. Washing of semen The pooled semen sample was diluted (1:5) with warm (30 ◦ C) Tris buffer and centrifuged at room temperature (24–27 ◦ C) for 10 min at 3000 × g. The clear supernatant was aspirated with the aid of a Pasteur pipette. Tris buffer was then added into the sediment to restore the initial volume during the first washing and centrifuged again. After the second washing, the extension of the sediment was done using a Tris extender. The volume of fresh semen prior to washing was taken into consideration for extension of sediment at the rate of 1:10. The same washing method was used for processing in Methods I and II. 2.2. Holding of semen The pooled semen sample was placed in a 5ml plastic tube and properly sealed and stored in a biochemical oxygen demand (BOD) incubator for incubation or holding of the semen at 24 ◦ C for different periods (1, 3 and 5 h)—before processing of the undiluted semen and before preservation of the washed and extended semen. 2.3. Extension of semen A Tris extender (Foote, 1970) modified by Deka and Rao (1986) was used for dilution of semen (Table 1). All the components of the extender, except egg yolk were mixed and the pH adjusted to 6.8 using a 5% citric acid solution. The egg yolk was added just prior to the Table 1 Composition of Tris extender used in the dilution of buck semen Components
Quantity
Tris (hydroxymethylaminomethane) (g) Citric acid (g) Fructose (g) Egg yolk (ml) Distilled water (ml) Penicillin (IU) Streptomycin (g)
2.422 1.34 1.0 20 80 80000 100000
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
collection of semen. The dilution was done with Tris extender at 30 ◦ C for undiluted semen and at 24 ◦ C for the incubated semen.
3. Processing methods 3.1. Method I (holding after washing) The fresh-pooled semen was washed twice using an isotonic buffer (Tris buffer) and then extended with a Tris–citric acid–fructose–egg yolk extender (Foote, 1970; Deka and Rao, 1986) at the rate of 1:10—taking into consideration the initial volume of the fresh semen sample used for washing. The extended semen was then stored at 24 ◦ C for 5 h in a biochemical oxygen demand incubator. Aliquots of incubated semen were removed at 0 h (without holding), 1, 3 and 5 h following holding and preserved at 5 ◦ C for 72 h. 3.2. Method II (washing after holding) The fresh-pooled semen sample was stored for 5 h at 24 ◦ C in a BOD incubator. Thereafter, the seminal plasma was removed by washing the aliquots twice at 0 (without holding), 1, 3 and 5 h following holding. Washed semen was then extended (1:10) with a Tris extender and preserved at 5 ◦ C for 72 h. 3.3. Method III (holding of undiluted semen) The fresh-pooled semen sample was stored at 24 ◦ C for 5 h in a BOD incubator. The aliquots of incubated semen were removed at 0 h (without holding), 1, 3 and 5 h following holding and extended at the rate of 1:10 with a Tris extender. The extended semen samples were then preserved at 5 ◦ C for 72 h. Semen samples were evaluated for individual progressive sperm motility following 0, 24, 48 and 72 h of preservation. The sperm motility (%) was evaluated by the conventional method as described by Zemjanis (1970). The determination of the % live sperm was performed using a nigrosin–eosin stain. Unstained spermatozoa were considered to be live and a total of 200 cells were evaluated to determine the % live spermatozoa (Blom, 1977). The incidence of intact acrosomes was also evaluated using a Giemsa stain as described by Watson (1975).
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3.4. Statistical analysis The data were analyzed using three-factor analysis of variance after transforming the percentages into arcsine values as per Snedecor and Cochran (1989). If a main effect or an interaction between main effects was significant, then, the differences between pairs of means were tested by the least significant difference (critical difference) test.
4. Results The mean sperm motility, % live sperm and intact acrosome in semen for the three different processing methods at different times of preservation at 5 ◦ C are set out in Tables 2–4. The sperm motility, live sperm count and intact acrosome at each stage of preservation for each time of holding were significantly (P < 0.01) higher following the use of Method I, compared to II and III (Tables 5 and 6). The critical difference test revealed that sperm motility, live sperm and incidence of intact acrosomes decreased significantly (P < 0.05) at each time of holding from 0 to 5 h and at each stage of preservation from 0 to 72 h (Tables 7 and 8). The sperm motility, live sperm count and intact acrosome declined progressively as the holding time at 24 ◦ C increased from 0 to 5 h in all the processing methods (Tables 2–4). The sperm motility of 85.8% at 0 h decreased to 71.7% after 5 h at 0 h of preservation with Method I. The corresponding values were 85.8 and 21.7% for Method II and 84.2 and 45.0% for Method III. After 72 h of storage, the motility was recorded as 66.7 and 49.2% for semen held for 0 and 5 h, using Method I. In Methods II and III, the respective values were 66.7 and 8.0%, and 42.5 and 25.8%, respectively. In Method I, the live sperm decreased from 89.7 to 81.3% as the duration of holding increased from 0 to 5 h, while in Method II % live sperm decreased from 89.7 to 34.8% and in Method III from 88.0 to 71.2% at 0 h preservation. Following 72 h of preservation, the corresponding values were 83.0 and 74.3% for Method I, 83.0 and 21.3% for Method II and 65.2 and 48.2% for Method III, respectively. The incidence of intact acrosomes (85.0% at 0 h) decreased to 75.8% as the holding time increased to 5 h for Method I and acrosomal integrity decreased from 85.0 to 59.7% in Method
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Table 2 The mean ± S.E. percentage motile sperm in Tris extender at different times of preservation at 5 ◦ C using different processing methods Preservation period (h)
Processing methods of semen
Overall mean
Method I holding after washing (h) 0
1
85.8 78.3 72.5 66.7
0.3 0.7 0.7 0.4
Overall mean
75.8 ± 0.7
82.0 74.2 68.3 60.8
± ± ± ±
0.3 0.3 0.4 0.1
71.3 ± 0.7
77.0 70.0 61.7 54.2
5 ± ± ± ±
0.4 0.5 0.9 1.4
65.7 ± 0.9
71.7 64.2 57.5 49.2
0 ± ± ± ±
0.4 1.1 1.3 1.8
60.6 ± 0.9
85.8 78.3 72.5 66.7
1 ± ± ± ±
0.3 0.7 0.7 0.4
75.8 ± 0.7
48.3 39.2 32.5 25.0
Method III holding of undiluted semen (h)
3 ± ± ± ±
0.4 0.6 0.7 0.5
36.3 ± 0.8
30.8 22.5 17.5 13.3
5 ± ± ± ±
1.8 1.6 1.3 2.5
21.0 ± 0.9
21.7 14.2 11.3 8.0
0 ± ± ± ±
1.6 1.6 1.5 1.3
13.8 ± 0.8
84.2 74.2 59.8 42.5
1 ± ± ± ±
0.6 0.6 1.5 3.7
65.2 ± 0.8
77.5 66.7 51.7 36.7
3 ± ± ± ±
0.7 0.7 1.1 3.2
58.1 ± 0.8
66.8 60.2 46.7 35.8
5 ± ± ± ±
0.5 0.8 0.4 0.6
52.4 ± 0.5
45.0 37.8 33.8 25.8
± ± ± ±
0.5 0.5 0.5 0.8
64.7 56.6 48.8 40.4
± ± ± ±
0.3 0.3 0.3 0.3
35.6 ± 0.3
Table 3 The mean ± S.E. percentage live sperm in Tris extender at different times of preservation at 5 ◦ C using different processing methods Preservation period (h)
Processing methods of semen
Overall mean
Method I holding after washing (h) 0
1 ± ± ± ±
0 24 48 72
89.7 86.8 83.8 83.0
0.3 0.8 0.4 0.4
Overall mean
85.8 ± 0.3
86.8 85.2 82.5 81.7
Method II washing after holding (h)
3 ± ± ± ±
0.6 0.4 0.5 0.5
84.0 ± 0.3
84.8 81.8 79.2 78.3
5 ± ± ± ±
0.4 0.4 0.5 0.5
81.0 ± .0.3
81.3 79.3 77.2 74.3
0 ± ± ± ±
0.4 0.5 0.3 1.1
78.0 ± 0.4
89.7 86.8 83.8 83.0
1 ± ± ± ±
0.3 0.8 0.4 0.4
85.8 ± 0.3
62.3 48.0 47.5 41.0
Method III holding of undiluted semen (h)
3 ± ± ± ±
0.4 0.7 0.1 1.6
49.7 ± 0.8
48.7 31.2 29.5 27.5
5 ± ± ± ±
1.8 1.8 2.2 1.7
34.2 ± 1.1
34.8 26.8 23.3 21.3
0 ± ± ± ±
1.4 3.3 3.0 2.7
26.6 ± 1.3
88.0 86.3 83.0 65.2
1 ± ± ± ±
0.5 0.5 0.5 5.4
80.6 ± 0.7
84.0 79.7 70.7 57.0
3 ± ± ± ±
0.7 0.4 0.2 4.7
72.8 ± 0.7
76.8 74.2 64.5 55.8
5 ± ± ± ±
0.4 0.2 0.4 0.7
67.8 ± 0.7
71.2 65.5 58.7 48.2
± ± ± ±
0.7 0.5 0.5 0.3
60.9 ± 0.8
74.9 69.3 65.3 59.7
± ± ± ±
0.2 0.3 0.3 0.3
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
± ± ± ±
0 24 48 72
Method II washing after holding (h)
3
68.3 ± 0.3 73.1 ± 0.2 78.5 ± 0.2 80.6 ± 0.3 49.4 ± 0.7 54.9 ± 1.0 58.8 ± 0.9 82.1 ± 0.4 68.9 ± 0.8 71.1 ± 0.8 82.1 ± 0.4 Overall mean
75.9 ± 0.5
77.7 71.5 63.5 60.5 0.4 0.5 0.5 0.5 ± ± ± ± 79.5 74.0 71.7 67.2
3
0.4 0.5 0.6 0.3 ± ± ± ± 85.5 80.0 76.7 71.7
1
0.4 0.7 1.0 0.7 ± ± ± ± 0
88.3 82.5 78.2 73.3 65.2 64.0 58.7 47.5 75.8 68.8 67.2 63.8 76.7 71.3 69.3 67.2 77.8 76.8 76.0 72.8 ± ± ± ±
0.7 1.0 0.6 0.3 85.0 83.2 81.0 79.3 0 24 48 72
1
± ± ± ±
1.5 1.1 0.6 0.4
3
± ± ± ±
1.6 2.1 1.9 0.9
5
± ± ± ±
1.9 0.9 1.5 1.5
85.0 83.2 81.0 79.3
± ± ± ±
0.9 0.1 0.6 0.3
1
± ± ± ±
1.3 1.2 2.2 0.4
61.8 60.7 56.8 40.3
± ± ± ±
2.0 1.5 0.8 0.7
59.7 53.3 43.3 41.3
± ± ± ±
1.1 1.1 0.5 0.4
Method III holding of undiluted semen (h)
5 0 0
3
Method II washing after holding (h) Method I holding after washing (h)
Processing methods of semen
Preservation period (h)
Table 4 The mean ± S.E. percentage intact acrosomes in Tris extender at different times of preservation at 5 ◦ C using different processing methods
5
± ± ± ±
0.3 1.1 0.8 0.8
76.5 72.4 68.6 63.7
± ± ± ±
0.2 0.2 0.2 0.2
Overall mean
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
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II and from 88.3 to 77.7% in Method III, at 0 h preservation. Following 72 h preservation, the percentage intact acrosomes was 79.3% at 0 h of holding and 63.8% for semen kept up to 5 h in Method I, with the corresponding values being 79.3 and 41.3% for Method II and 73.3 and 60.5% in Method III.
5. Discussion The sperm motility at each stage of preservation for each holding time was observed to be higher in Method I (holding after washing). The lower sperm motility observed in Methods II and III may be due to insufficient substrate in the undiluted semen, resulting in the death of a large proportion of sperm during holding. The fructose available in semen has been reported to support sperm metabolism for approximately 30 min (Mann and Lutwak-Mann, 1981). The detrimental effect of washing after holding in Method II was evident at 0 h of preservation and was similarly reflected during subsequent preservation at 5 ◦ C. The holding of semen causes considerable cell damage (Lenz et al., 1977; Robertson et al., 1989) and there was further damage to sperm due to double washing after holding for Method II resulting in lower sperm motility. Sperm motility was higher at 0 h (without holding) than when the semen was kept for 1, 3 and 5 h after washing and dilution (Method I). The loss of sperm motility has been reported during incubation of diluted bovine semen in 2.9% sodium citrate solution at 37 ◦ C for 8 h (Brown and Senger, 1980). Similarly, the sperm motility was reported to markedly decrease during the holding of ram semen for 4 h at 30 ◦ C (Robertson et al., 1989) and washed bovine sperm for 6 h at 20 and 35 ◦ C (Soderquist, 1991). The decline in sperm motility during holding of semen at 24 ◦ C in the present study may be due to the higher metabolic activity of sperm at this temperature, causing a reduction in available nutrients and accumulation of lactic acid. The decrease in semen quality, with the increase in holding time recorded during subsequent preservation (24, 48 and 72 h) at 5 ◦ C, was a reflection of the initial decline in semen quality immediately after holding. It was further observed that the washing of sperm using isotonic Tris buffer after holding (Method II), resulted in the formation of a pellet at the bottom of the centrifuge tube, with the size of the pellet increasing with
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R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
Table 5 Analysis of variance of the percentage motile sperm, live sperm and intact acrosomes, in Tris extender at different times of preservation at 5 ◦ C using different processing methods Source of variation
Processing methods (PM) Holding times (HT) Preservation periods (PP) PM × HT PM × PP HT × PP PM × HT × PP Error * ** a
d.f.
2 3 3 6 6 9 18 240
Mean squares Sperm motility
Live sperm
Intact acrosome
9515.75**
10528.30**
6494.32** 3363.16** 1395.06** 115.44** 30.20a 23.85a 28.49
4791.25** 1375.69** 1432.99** 206.61** 13.61a 14.95a 46.49
2158.07** 2112.64** 904.84** 280.61** 39.67* 15.42a 11.72a 15.83
P < 0.05. P < 0.01. Non-significant.
Table 6 Critical difference test in mean sperm motility, % live sperm and intact acrosomes, irrespective of the holding time and preservation period Processing methods
Sperm motility (%)
Live sperm (%)
Intact acrosome (%)
I II III
68.38a ± 0.11 36.73c ± 0.27 52.82b ± 0.19
82.24a ± 0.05 49.08c ± 0.27 70.54b ± 0.16
74.5a ± 0.10 61.32b ± 0.16 75.10a ± 0.08
Means (a–c) in columns with different superscripts differ significantly (P < 0.05).
an increase in holding time. On the other hand, washing of semen using a Tris buffer before holding (Method I) did not cause the formation of pellet. The pellet formation in Method II may be related to cell membrane damage causing stickiness of the cell.
The live sperm count immediately after holding (0 h) in the processing methods decreased with an increase in holding time at 24 ◦ C from 0 to 5 h. A similar decline in the % live sperm was also reported during incubation of ram semen at 30 ◦ C (Quinn et al., 1968; Robertson et
Table 7 Critical difference test in mean sperm motility, % live sperm and intact acrosomes, irrespsective of the processing method and preservation period Holding time (h)
Sperm motility (%)
Live sperm (%)
Intact acrosome (%)
0 1 3 5
± 0.19 55.24b ± 0.27 46.38c ± 0.30 36.68d ± 0.30
± 0.15 68.86b ± 0.25 61.03c ± 0.31 55.17d ± .33
81.61a 71.06b 66.38c 62.21d
72.28a
84.10a
± ± ± ±
0.08 0.16 0.17 0.18
Means (a–d) in columns with different superscripts differ significantly (P < 0.05). Table 8 Critical difference test in mean sperm motility, % live sperm and intact acrosomes, irrespective of the processing method and holding time Preservation periods (h) 0 24 48 72
Sperm motility (%)
Live sperm (%)
Intact acrosome (%)
± 0.31 56.64b ± 0.31 48.82c ± 0.29 40.39d ± 0.30
± 0.24 69.31b ± 0.31 65.31c ± 0.30 59.69d ± 0.34
76.50a 72.44b 68.61c 63.69d
64.72a
Means (a–d) in columns with different superscripts differ significantly (P < 0.05).
74.85a
± ± ± ±
0.16 0.15 0.17 0.19
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
al., 1989) and bull semen at 37 ◦ C (Lenz et al., 1977). The significantly (P < 0.05) lower percentage intact acrosomes recorded in Method II compared to Method I, could be attributed to the washing after holding. Both incubation (holding) and washing of sperm are known to cause damage to the plasma membrane and the acrosome (Jones and Holt, 1974; Lenz et al., 1977; Robertson, 1989). The lower percentage of sperm with intact acrosomes in Method II may be due to the fact that, in this method, spermatozoa that had already suffered damage due to incubation were further washed twice. The highest sperm motility, live sperm and intact acrosomes at 5 ◦ C up to 72 h of preservation maintained in washed semen not subjected to holding. The beneficial effect of removal of seminal plasma on the quality of goat semen during preservation observed in the present study was in agreement with the earlier reports (Memon et al., 1985; Deka and Rao, 1986; Misra et al., 1993). This improvement in the quality of washed semen recorded during preservation for a period of up to 72 h may be due to the removal of the egg yolkcoagulating enzyme present in the goat semen secreted by the bulbo-urethral glands, which caused coagulation of egg yolk present in the diluent (Roy, 1957). Literature revealed no study on the effect of holding of buck semen, with or without seminal plasma, on the quality of semen at different hours of preservation at 5 ◦ C. Hence, the combined effect (holding with washing) on quality of goat semen could not be compared. The present study demonstrated that the quality of goat semen during preservation at 5 ◦ C could not be improved by holding it at 24 ◦ C prior to preservation—as is reported to occur in boar semen (Pursel et al., 1973; Tamuli, 1993; Nath et al., 1994).
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Graham, E.F., Rajaraman, A.H.J., Schmehl, M.K.L., Makilaurila, M., Bower, R.E., 1971. Preliminary report on procedure and rationale for freezing of boar semen. A. I. Digest. 19 (1), 12–14 (Anim. Breed., Abstract 39, 3694). Irritani, A., Nishikawa, Y., Fukuhara, R., 1961. Studies on the egg yolk coagulating factor in goat semen. In: Proceedings of the Silver Jubilee, The Laboratory of Animal Husbandary, College of AGRI, Koyata, Japan (Anim. Breed., Abstract 32, 2143). Jones, R.C., Holt, W.V., 1974. The effect of washing on the ultra structure and cytochemistry of ram spermatozoa. J. Reprod. Fertil. 41 (1), 159–167. Lenz, R.W., Graves, C.N., Lodge, J.R., 1977. Influence of incubation in seminal plasma on subsequent metabolic and morphological characteristics of bovine spermatozoa. Theriogenology 7 (5), 265–276. Mann, T., Lutwak-Mann, C., 1981. Male reproductive function and semen. Springer, Berlin (cited by Robertson, L., Plummer, J.M., Watson, P.F., 1989. Anim. Reprod. Sci. 18, 285–299). Memon, M.A., Bretzlaff, K.N., Ott, R.S., 1985. Effect of washing on motility and acrosome morphology of frozen–thawed goat spermatozoa. Am. J. Vet. Res. 46 (2), 473–475. Misra, D.N., Deka, B.C., Borgohain, B.N., 1993. Effect of washing on the quality of goat semen during preservation at +5 ◦ C. Indian J. Anim. Reprod. 14 (1), 49–50. Nath, M.C., Deka, B.C., Borgohain, B.N., 1994. Effect of holding time on quality of frozen boar semen. Indian Vet. J. 71 (3), 250–252. Pursel, V.G., Schulman, L.L., Johnson, L.A., 1973. Effect of holding time on storage of boar spermatozoa during preservation at +5 ◦ C. J. Anim. Sci. 37 (3), 785–789. Quinn, P.J., Salamon, S., White, I.G., 1968. The effect of cold shock and deep freezing on ram spermatozoa collected by electrical stimulation and by an artificial vagina. Aust. J. Agric. Sci. 19, 119–128. Robertson, L., Plummer, J.M., Watson, P.F., 1989. Acquired resistance to cold shock in ram spermatozoa revealed by microscopy and a calcium radioassay. Anim. Reprod. Sci. 18, 286– 292. Roy, A., 1957. Egg yolk coagulating enzyme in the semen and Cowper’s glands of the goat. Nature (London) 179, 318–319 (Anim. Breed., Abstract 25, 799). Sahni, K.L., Roy, A., 1972. A note on the application of cold shock test for determining the resistance (quality) of sheep and goat spermatozoa. Indian J. Anim. Sci. 42, P198–P501. Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods, eighth ed. Iowa University Press, Ames, IA, USA. Soderquist, L., 1991. ATP content and sperm motility of extended bovine semen under different storage conditions. Acta Vet. Scand. 32, 511–518 (Anim. Breed., Abstract 61, 3055). Tamuli, M.K., 1993. Studies on certain aspects of the development of resistance to cold shock in boar spermatozoa. Ph.D. Thesis, Royal Veterinary College, University of London. Watson, P.F., 1975. Use of Giemsa stain to detect changes in acrosomes of frozen ram spermatozoa. Vet. Rec. 97, 12–15. Zemjanis, R., 1970. Diagnostic and Therapeutic Techniques in Animal Reproduction, second ed. Williams and Wilkins Co., Baltimore.
Effect of holding and washing on the quality of goat semen Rafiqul Islam ∗ , K. Ahmed, B.C. Deka Department of Gynaecology, Obstetrics and A.I., College of Veterinary Sciences, Assam Agricultural University, Khanapara, Guwahati 781022, Assam, India Received 14 October 2003; received in revised form 21 June 2005; accepted 21 June 2005 Available online 12 September 2005
Abstract A total of 18 pooled semen samples collected from 4 healthy crossbred (Beetal × Assam Local) bucks, twice weekly for a period of 12 weeks, were processed via three processing methods, viz. Method I (holding after washing: semen sample was washed twice, extended, held for 0, 1, 3 and 5 h and preserved), Method II (washing after holding: semen sample held for 0, 1, 3 and 5 h, washed twice, extended and preserved) and Method III (holding of undiluted semen: semen sample held for 0, 1, 3 and 5 h, extended and preserved). An isotonic Tris buffer was used to wash the spermatozoa with the aid of centrifugation at 3000 rotations per minute (rpm) for 10 min. A Tris–citric acid–fructose–egg yolk extender was used to extend the semen processed with all three processing methods and preserved at 5 ◦ C up to 72 h. Holding of semen at 24 ◦ C in each processing method caused deterioration of the semen quality during preservation, as the holding time increased from 0 (without holding), 1, 3 and 5 h. The initial sperm motility declined from 85.8 to 71.7% in Method I, from 85.8 to 21.7% in Method II and from 84.2 to 45.0% in Method III, as the holding time increased from 0 to 5 h. The corresponding values were 66.7 and 49.2% for Method I, 66.7 and 8.0% for Method II and 42.5 and 25.8% for Method III, following 72 h of preservation. The percentage of live sperm and intact acrosomes also showed a similar decreasing trend as the holding time increased from 0 to 5 h in all processing methods as revealed in percentage of sperm motility. Washing of spermatozoa without a holding period maintained significantly higher sperm motility (66.7%), live sperm counts (83.0%) and incidence of intact acrosomes (79.3%) following 72 h at preservation, compared to the corresponding values (42.5, 65.2 and 73.3% for sperm motility, live sperm counts and intact acrosomes, respectively) recorded in unwashed semen without a holding period. The values recorded for washed semen without holding were also significantly higher than for semen held for 1, 3 and 5 h in all the three processing methods following 72 h of preservation. This study revealed that the holding of semen caused deterioration of the spermatozoa, whereas, washing without holding had a beneficial effect on the viability of spermatozoa in maintaining its storage ability for up to 72 h of preservation at refrigeration temperature (5 ◦ C). © 2005 Published by Elsevier B.V. Keywords: Goat semen; Holding time; Seminal plasma removal; Preservation quality
∗
Corresponding author. Present address: Division of Animal Reproduction, Gynaecology and Obstetrics, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology (K), Shuhama, Alusteng, Srinagar 190006, Kashmir, India. Tel.: +91 194 2262208/2262209 (o); fax: +91 194 2262207/2262210. E-mail address: [email protected] (R. Islam). 0921-4488/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2005.06.034
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1. Introduction Goat spermatozoa are susceptible to cold shock, which causes the death of numerous spermatozoa during processing for preservation at lower temperature, although the degree of susceptibility is less than that experienced in boar spermatozoa (Sahni and Roy, 1972). Holding, a procedure of retaining or incubation of semen at different room temperatures (18–30 ◦ C) for a few hours before or after processing in boar semen prior to preservation, is generally beneficial (Graham et al., 1971; Pursel et al., 1973; Tamuli, 1993; Nath et al., 1994). Thus, it may be expected that a similar procedure may have some beneficial effect on goat spermatozoa and help sperms to develop a possible resistance against cold shock. However, there is no information currently available on the effect of the holding on the quality of goat semen during preservation at refrigeration temperature (5 ◦ C). An egg yolk-coagulating enzyme is present in the seminal plasma of goat semen, which has limited the use of egg yolk containing extenders for the preservation of goat semen (Roy, 1957). Removal of the seminal plasma by washing the spermatozoa with an isotonic buffer has been reported to improve the quality of goat semen during preservation at various temperatures (Irritani et al., 1961; Deka and Rao, 1986; Misra et al., 1993). However, the effect of the removal of seminal plasma on the quality of goat semen during preservation following holding has not been reported. This study was performed to determine the effect of holding, with or without seminal plasma, on the quality of goat semen for preservation at 5 ◦ C.
2. Materials and methods Four healthy crossbred bucks (Beetal × Assam Local) approximately, 2.5 years of age were selected from the Goat Research Station, Assam Agricultural University, Burnihat, Assam, India. The animals were maintained under standard managemental conditions practiced on the farm. Semen samples were collected twice weekly in the morning from each buck with the aid of the artificial vagina for a period of 12 weeks. Immediately after collection, each ejaculate was evaluated for mass motility (based on the numerical scale 0–4) and initial motility as per the method described by
Zemjanis (1970). Semen samples showing +3 or more mass motility and 80% or more initial motility were pooled. A total of 18 pooled semen samples were used for the study. 2.1. Washing of semen The pooled semen sample was diluted (1:5) with warm (30 ◦ C) Tris buffer and centrifuged at room temperature (24–27 ◦ C) for 10 min at 3000 × g. The clear supernatant was aspirated with the aid of a Pasteur pipette. Tris buffer was then added into the sediment to restore the initial volume during the first washing and centrifuged again. After the second washing, the extension of the sediment was done using a Tris extender. The volume of fresh semen prior to washing was taken into consideration for extension of sediment at the rate of 1:10. The same washing method was used for processing in Methods I and II. 2.2. Holding of semen The pooled semen sample was placed in a 5ml plastic tube and properly sealed and stored in a biochemical oxygen demand (BOD) incubator for incubation or holding of the semen at 24 ◦ C for different periods (1, 3 and 5 h)—before processing of the undiluted semen and before preservation of the washed and extended semen. 2.3. Extension of semen A Tris extender (Foote, 1970) modified by Deka and Rao (1986) was used for dilution of semen (Table 1). All the components of the extender, except egg yolk were mixed and the pH adjusted to 6.8 using a 5% citric acid solution. The egg yolk was added just prior to the Table 1 Composition of Tris extender used in the dilution of buck semen Components
Quantity
Tris (hydroxymethylaminomethane) (g) Citric acid (g) Fructose (g) Egg yolk (ml) Distilled water (ml) Penicillin (IU) Streptomycin (g)
2.422 1.34 1.0 20 80 80000 100000
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
collection of semen. The dilution was done with Tris extender at 30 ◦ C for undiluted semen and at 24 ◦ C for the incubated semen.
3. Processing methods 3.1. Method I (holding after washing) The fresh-pooled semen was washed twice using an isotonic buffer (Tris buffer) and then extended with a Tris–citric acid–fructose–egg yolk extender (Foote, 1970; Deka and Rao, 1986) at the rate of 1:10—taking into consideration the initial volume of the fresh semen sample used for washing. The extended semen was then stored at 24 ◦ C for 5 h in a biochemical oxygen demand incubator. Aliquots of incubated semen were removed at 0 h (without holding), 1, 3 and 5 h following holding and preserved at 5 ◦ C for 72 h. 3.2. Method II (washing after holding) The fresh-pooled semen sample was stored for 5 h at 24 ◦ C in a BOD incubator. Thereafter, the seminal plasma was removed by washing the aliquots twice at 0 (without holding), 1, 3 and 5 h following holding. Washed semen was then extended (1:10) with a Tris extender and preserved at 5 ◦ C for 72 h. 3.3. Method III (holding of undiluted semen) The fresh-pooled semen sample was stored at 24 ◦ C for 5 h in a BOD incubator. The aliquots of incubated semen were removed at 0 h (without holding), 1, 3 and 5 h following holding and extended at the rate of 1:10 with a Tris extender. The extended semen samples were then preserved at 5 ◦ C for 72 h. Semen samples were evaluated for individual progressive sperm motility following 0, 24, 48 and 72 h of preservation. The sperm motility (%) was evaluated by the conventional method as described by Zemjanis (1970). The determination of the % live sperm was performed using a nigrosin–eosin stain. Unstained spermatozoa were considered to be live and a total of 200 cells were evaluated to determine the % live spermatozoa (Blom, 1977). The incidence of intact acrosomes was also evaluated using a Giemsa stain as described by Watson (1975).
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3.4. Statistical analysis The data were analyzed using three-factor analysis of variance after transforming the percentages into arcsine values as per Snedecor and Cochran (1989). If a main effect or an interaction between main effects was significant, then, the differences between pairs of means were tested by the least significant difference (critical difference) test.
4. Results The mean sperm motility, % live sperm and intact acrosome in semen for the three different processing methods at different times of preservation at 5 ◦ C are set out in Tables 2–4. The sperm motility, live sperm count and intact acrosome at each stage of preservation for each time of holding were significantly (P < 0.01) higher following the use of Method I, compared to II and III (Tables 5 and 6). The critical difference test revealed that sperm motility, live sperm and incidence of intact acrosomes decreased significantly (P < 0.05) at each time of holding from 0 to 5 h and at each stage of preservation from 0 to 72 h (Tables 7 and 8). The sperm motility, live sperm count and intact acrosome declined progressively as the holding time at 24 ◦ C increased from 0 to 5 h in all the processing methods (Tables 2–4). The sperm motility of 85.8% at 0 h decreased to 71.7% after 5 h at 0 h of preservation with Method I. The corresponding values were 85.8 and 21.7% for Method II and 84.2 and 45.0% for Method III. After 72 h of storage, the motility was recorded as 66.7 and 49.2% for semen held for 0 and 5 h, using Method I. In Methods II and III, the respective values were 66.7 and 8.0%, and 42.5 and 25.8%, respectively. In Method I, the live sperm decreased from 89.7 to 81.3% as the duration of holding increased from 0 to 5 h, while in Method II % live sperm decreased from 89.7 to 34.8% and in Method III from 88.0 to 71.2% at 0 h preservation. Following 72 h of preservation, the corresponding values were 83.0 and 74.3% for Method I, 83.0 and 21.3% for Method II and 65.2 and 48.2% for Method III, respectively. The incidence of intact acrosomes (85.0% at 0 h) decreased to 75.8% as the holding time increased to 5 h for Method I and acrosomal integrity decreased from 85.0 to 59.7% in Method
54
Table 2 The mean ± S.E. percentage motile sperm in Tris extender at different times of preservation at 5 ◦ C using different processing methods Preservation period (h)
Processing methods of semen
Overall mean
Method I holding after washing (h) 0
1
85.8 78.3 72.5 66.7
0.3 0.7 0.7 0.4
Overall mean
75.8 ± 0.7
82.0 74.2 68.3 60.8
± ± ± ±
0.3 0.3 0.4 0.1
71.3 ± 0.7
77.0 70.0 61.7 54.2
5 ± ± ± ±
0.4 0.5 0.9 1.4
65.7 ± 0.9
71.7 64.2 57.5 49.2
0 ± ± ± ±
0.4 1.1 1.3 1.8
60.6 ± 0.9
85.8 78.3 72.5 66.7
1 ± ± ± ±
0.3 0.7 0.7 0.4
75.8 ± 0.7
48.3 39.2 32.5 25.0
Method III holding of undiluted semen (h)
3 ± ± ± ±
0.4 0.6 0.7 0.5
36.3 ± 0.8
30.8 22.5 17.5 13.3
5 ± ± ± ±
1.8 1.6 1.3 2.5
21.0 ± 0.9
21.7 14.2 11.3 8.0
0 ± ± ± ±
1.6 1.6 1.5 1.3
13.8 ± 0.8
84.2 74.2 59.8 42.5
1 ± ± ± ±
0.6 0.6 1.5 3.7
65.2 ± 0.8
77.5 66.7 51.7 36.7
3 ± ± ± ±
0.7 0.7 1.1 3.2
58.1 ± 0.8
66.8 60.2 46.7 35.8
5 ± ± ± ±
0.5 0.8 0.4 0.6
52.4 ± 0.5
45.0 37.8 33.8 25.8
± ± ± ±
0.5 0.5 0.5 0.8
64.7 56.6 48.8 40.4
± ± ± ±
0.3 0.3 0.3 0.3
35.6 ± 0.3
Table 3 The mean ± S.E. percentage live sperm in Tris extender at different times of preservation at 5 ◦ C using different processing methods Preservation period (h)
Processing methods of semen
Overall mean
Method I holding after washing (h) 0
1 ± ± ± ±
0 24 48 72
89.7 86.8 83.8 83.0
0.3 0.8 0.4 0.4
Overall mean
85.8 ± 0.3
86.8 85.2 82.5 81.7
Method II washing after holding (h)
3 ± ± ± ±
0.6 0.4 0.5 0.5
84.0 ± 0.3
84.8 81.8 79.2 78.3
5 ± ± ± ±
0.4 0.4 0.5 0.5
81.0 ± .0.3
81.3 79.3 77.2 74.3
0 ± ± ± ±
0.4 0.5 0.3 1.1
78.0 ± 0.4
89.7 86.8 83.8 83.0
1 ± ± ± ±
0.3 0.8 0.4 0.4
85.8 ± 0.3
62.3 48.0 47.5 41.0
Method III holding of undiluted semen (h)
3 ± ± ± ±
0.4 0.7 0.1 1.6
49.7 ± 0.8
48.7 31.2 29.5 27.5
5 ± ± ± ±
1.8 1.8 2.2 1.7
34.2 ± 1.1
34.8 26.8 23.3 21.3
0 ± ± ± ±
1.4 3.3 3.0 2.7
26.6 ± 1.3
88.0 86.3 83.0 65.2
1 ± ± ± ±
0.5 0.5 0.5 5.4
80.6 ± 0.7
84.0 79.7 70.7 57.0
3 ± ± ± ±
0.7 0.4 0.2 4.7
72.8 ± 0.7
76.8 74.2 64.5 55.8
5 ± ± ± ±
0.4 0.2 0.4 0.7
67.8 ± 0.7
71.2 65.5 58.7 48.2
± ± ± ±
0.7 0.5 0.5 0.3
60.9 ± 0.8
74.9 69.3 65.3 59.7
± ± ± ±
0.2 0.3 0.3 0.3
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
± ± ± ±
0 24 48 72
Method II washing after holding (h)
3
68.3 ± 0.3 73.1 ± 0.2 78.5 ± 0.2 80.6 ± 0.3 49.4 ± 0.7 54.9 ± 1.0 58.8 ± 0.9 82.1 ± 0.4 68.9 ± 0.8 71.1 ± 0.8 82.1 ± 0.4 Overall mean
75.9 ± 0.5
77.7 71.5 63.5 60.5 0.4 0.5 0.5 0.5 ± ± ± ± 79.5 74.0 71.7 67.2
3
0.4 0.5 0.6 0.3 ± ± ± ± 85.5 80.0 76.7 71.7
1
0.4 0.7 1.0 0.7 ± ± ± ± 0
88.3 82.5 78.2 73.3 65.2 64.0 58.7 47.5 75.8 68.8 67.2 63.8 76.7 71.3 69.3 67.2 77.8 76.8 76.0 72.8 ± ± ± ±
0.7 1.0 0.6 0.3 85.0 83.2 81.0 79.3 0 24 48 72
1
± ± ± ±
1.5 1.1 0.6 0.4
3
± ± ± ±
1.6 2.1 1.9 0.9
5
± ± ± ±
1.9 0.9 1.5 1.5
85.0 83.2 81.0 79.3
± ± ± ±
0.9 0.1 0.6 0.3
1
± ± ± ±
1.3 1.2 2.2 0.4
61.8 60.7 56.8 40.3
± ± ± ±
2.0 1.5 0.8 0.7
59.7 53.3 43.3 41.3
± ± ± ±
1.1 1.1 0.5 0.4
Method III holding of undiluted semen (h)
5 0 0
3
Method II washing after holding (h) Method I holding after washing (h)
Processing methods of semen
Preservation period (h)
Table 4 The mean ± S.E. percentage intact acrosomes in Tris extender at different times of preservation at 5 ◦ C using different processing methods
5
± ± ± ±
0.3 1.1 0.8 0.8
76.5 72.4 68.6 63.7
± ± ± ±
0.2 0.2 0.2 0.2
Overall mean
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
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II and from 88.3 to 77.7% in Method III, at 0 h preservation. Following 72 h preservation, the percentage intact acrosomes was 79.3% at 0 h of holding and 63.8% for semen kept up to 5 h in Method I, with the corresponding values being 79.3 and 41.3% for Method II and 73.3 and 60.5% in Method III.
5. Discussion The sperm motility at each stage of preservation for each holding time was observed to be higher in Method I (holding after washing). The lower sperm motility observed in Methods II and III may be due to insufficient substrate in the undiluted semen, resulting in the death of a large proportion of sperm during holding. The fructose available in semen has been reported to support sperm metabolism for approximately 30 min (Mann and Lutwak-Mann, 1981). The detrimental effect of washing after holding in Method II was evident at 0 h of preservation and was similarly reflected during subsequent preservation at 5 ◦ C. The holding of semen causes considerable cell damage (Lenz et al., 1977; Robertson et al., 1989) and there was further damage to sperm due to double washing after holding for Method II resulting in lower sperm motility. Sperm motility was higher at 0 h (without holding) than when the semen was kept for 1, 3 and 5 h after washing and dilution (Method I). The loss of sperm motility has been reported during incubation of diluted bovine semen in 2.9% sodium citrate solution at 37 ◦ C for 8 h (Brown and Senger, 1980). Similarly, the sperm motility was reported to markedly decrease during the holding of ram semen for 4 h at 30 ◦ C (Robertson et al., 1989) and washed bovine sperm for 6 h at 20 and 35 ◦ C (Soderquist, 1991). The decline in sperm motility during holding of semen at 24 ◦ C in the present study may be due to the higher metabolic activity of sperm at this temperature, causing a reduction in available nutrients and accumulation of lactic acid. The decrease in semen quality, with the increase in holding time recorded during subsequent preservation (24, 48 and 72 h) at 5 ◦ C, was a reflection of the initial decline in semen quality immediately after holding. It was further observed that the washing of sperm using isotonic Tris buffer after holding (Method II), resulted in the formation of a pellet at the bottom of the centrifuge tube, with the size of the pellet increasing with
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R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
Table 5 Analysis of variance of the percentage motile sperm, live sperm and intact acrosomes, in Tris extender at different times of preservation at 5 ◦ C using different processing methods Source of variation
Processing methods (PM) Holding times (HT) Preservation periods (PP) PM × HT PM × PP HT × PP PM × HT × PP Error * ** a
d.f.
2 3 3 6 6 9 18 240
Mean squares Sperm motility
Live sperm
Intact acrosome
9515.75**
10528.30**
6494.32** 3363.16** 1395.06** 115.44** 30.20a 23.85a 28.49
4791.25** 1375.69** 1432.99** 206.61** 13.61a 14.95a 46.49
2158.07** 2112.64** 904.84** 280.61** 39.67* 15.42a 11.72a 15.83
P < 0.05. P < 0.01. Non-significant.
Table 6 Critical difference test in mean sperm motility, % live sperm and intact acrosomes, irrespective of the holding time and preservation period Processing methods
Sperm motility (%)
Live sperm (%)
Intact acrosome (%)
I II III
68.38a ± 0.11 36.73c ± 0.27 52.82b ± 0.19
82.24a ± 0.05 49.08c ± 0.27 70.54b ± 0.16
74.5a ± 0.10 61.32b ± 0.16 75.10a ± 0.08
Means (a–c) in columns with different superscripts differ significantly (P < 0.05).
an increase in holding time. On the other hand, washing of semen using a Tris buffer before holding (Method I) did not cause the formation of pellet. The pellet formation in Method II may be related to cell membrane damage causing stickiness of the cell.
The live sperm count immediately after holding (0 h) in the processing methods decreased with an increase in holding time at 24 ◦ C from 0 to 5 h. A similar decline in the % live sperm was also reported during incubation of ram semen at 30 ◦ C (Quinn et al., 1968; Robertson et
Table 7 Critical difference test in mean sperm motility, % live sperm and intact acrosomes, irrespsective of the processing method and preservation period Holding time (h)
Sperm motility (%)
Live sperm (%)
Intact acrosome (%)
0 1 3 5
± 0.19 55.24b ± 0.27 46.38c ± 0.30 36.68d ± 0.30
± 0.15 68.86b ± 0.25 61.03c ± 0.31 55.17d ± .33
81.61a 71.06b 66.38c 62.21d
72.28a
84.10a
± ± ± ±
0.08 0.16 0.17 0.18
Means (a–d) in columns with different superscripts differ significantly (P < 0.05). Table 8 Critical difference test in mean sperm motility, % live sperm and intact acrosomes, irrespective of the processing method and holding time Preservation periods (h) 0 24 48 72
Sperm motility (%)
Live sperm (%)
Intact acrosome (%)
± 0.31 56.64b ± 0.31 48.82c ± 0.29 40.39d ± 0.30
± 0.24 69.31b ± 0.31 65.31c ± 0.30 59.69d ± 0.34
76.50a 72.44b 68.61c 63.69d
64.72a
Means (a–d) in columns with different superscripts differ significantly (P < 0.05).
74.85a
± ± ± ±
0.16 0.15 0.17 0.19
R. Islam et al. / Small Ruminant Research 66 (2006) 51–57
al., 1989) and bull semen at 37 ◦ C (Lenz et al., 1977). The significantly (P < 0.05) lower percentage intact acrosomes recorded in Method II compared to Method I, could be attributed to the washing after holding. Both incubation (holding) and washing of sperm are known to cause damage to the plasma membrane and the acrosome (Jones and Holt, 1974; Lenz et al., 1977; Robertson, 1989). The lower percentage of sperm with intact acrosomes in Method II may be due to the fact that, in this method, spermatozoa that had already suffered damage due to incubation were further washed twice. The highest sperm motility, live sperm and intact acrosomes at 5 ◦ C up to 72 h of preservation maintained in washed semen not subjected to holding. The beneficial effect of removal of seminal plasma on the quality of goat semen during preservation observed in the present study was in agreement with the earlier reports (Memon et al., 1985; Deka and Rao, 1986; Misra et al., 1993). This improvement in the quality of washed semen recorded during preservation for a period of up to 72 h may be due to the removal of the egg yolkcoagulating enzyme present in the goat semen secreted by the bulbo-urethral glands, which caused coagulation of egg yolk present in the diluent (Roy, 1957). Literature revealed no study on the effect of holding of buck semen, with or without seminal plasma, on the quality of semen at different hours of preservation at 5 ◦ C. Hence, the combined effect (holding with washing) on quality of goat semen could not be compared. The present study demonstrated that the quality of goat semen during preservation at 5 ◦ C could not be improved by holding it at 24 ◦ C prior to preservation—as is reported to occur in boar semen (Pursel et al., 1973; Tamuli, 1993; Nath et al., 1994).
References Blom, E., 1977. Sperm morphology with reference to bull infertility. In: Some Papers Contributed to the First All India Symposium on Animal Reproduction, Punjab Agricultural University, Ludhiana, India, pp. 66–81. Brown, D.V., Senger, P.L., 1980. Influence of homologous blood serum on motility and head-to-head agglutination in non motile ejaculated bovine spermatozoa. Biol. Reprod. 23 (2), 271–275. Deka, B.C., Rao, A.R., 1986. Motility of buck spermatozoa during preservation at 5 ◦ C with and without seminal plasma. Indian Vet. J. 63, 169–170. Foote, R.H., 1970. Fertility of bull semen at high extension rates in Tris buffered extenders. J. Dairy Sci. 53, 1475.
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