An osmotic resistance test for bovine semen

ANIMAL REPRODUCTION SCIENCE Animal Reproduction Science 36 (1994) 77-86

ELSEVIER

An osmotic resistance test for bovine semen S . G . R e v e l l *'a, R . A . M r o d e b aGenusFreezing Unit, Llanrhydd, Ruthin LL15 2UP, UK bMilk Marketing Board, Thames Ditton KT7 0EL, UK

(Accepted 8 September 1993 )

Abstract An osmotic resistance test (ORT) was developed for bull semen. Raw semen was incubated for 40-60 min at 35 °C in a fructose/sodium citrate solution with an osmolarity of 150 mOsm k g - 1. Semen processed in skim milk, egg yolk and glycerol diluent was mixed 1:4 with a similar solution at 100 mOsm kg -I. On examination with a phase contrast microscope ( × 400), cells with intact membranes were swollen. Some of these showed residual activity, i.e. a non-progressive flickering of the mid-piece. The proportion (%) of cells showing this activity was taken as the test result. Repeatability was 0.85 for raw semen and 0.93 for post-thaw. Over 401 ejaculates from 80 bulls (three straws per ejaculate), 51.2% of variation was due to bull, 33.7% to ejaculate within bull and 15.1% to straw within ejaculate. Five or six ejaculates from yearling Holstein/Friesian bulls were used for a fertility trial. Each was ORT tested post-thaw and two groups of ejaculates formed for each bull according to ORT result. Friesian/Holstein cows were inseminated and 49-day nonreturn rates (NRR) obtained for each group. Raw ORT correlated with post-thaw ORT (r=0.56), but not with NRR (r=0.08). Over a series of 48 bulls, the post-thaw ORT:NRR correlation was 0.37, but after removing bull effects a correlation of 0.79 was obtained. As the method is simple and rapid, it shows promise as a routine assessment technique in commercial AI laboratories.

1. Introduction D e s p i t e the w i d e s p r e a d use o f AI in cattle o v e r m a n y years, quality control r e m a i n s relatively crude. It is generally b a s e d on the m o r e o b v i o u s attributes o f *Corresponding author. 0378-4320/94/$07.00 © 1994 Elsevier Science Publishers B.V. All rights reserved SSD10378-4320 (93) 01279-H

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sperm, i.e. their motility and the proportion conforming to accepted standards of morphological normality. Such standards have to date proven to be very blunt instruments, with a very limited correlation with fertility (Bishop et al., 1954; Linford et al., 1976; Wood et al., 1986; Soderquist et al., 1991 ). The development of an in vitro test or tests capable of predicting fertility would be of great benefit to the industry. This paper reports the development of an osmotic resistance test (ORT) which shows a better correlation with fertility than current tests based solely on motility.

2. Materials and methods

The standard method for testing raw semen consisted of the incubation of 510 #1 of semen (depending on density) in 1 ml of test solution (osmotic strength 150 mOsm kg-1 ) for between 40 and 60 min at 35 oC. The solution consisted of fructose and trisodium citrate in glass-distilled or reverse osmosis/ion exchange purified water (Elgastat Option 4, Elga, High Wycombe, UK) (Table 1 ). The osmolarity of each batch was measured using a Knauer osmometer. Spermatozoa whose plasma membrane is intact swell in response to this treatment; a process seen as bending, coiling or shortening of the tail (Fig. 1 ). Some of these swollen cells continue to show activity. This is non-progressive on account of the tail deformity and normally takes the form of a flickering of the midpiece. Following incubation, the preparation was mixed well. A 10/A drop was transferred to a warm, clean microscope slide and covered with a 22 m m X 2 6 mm coverslip. This preparation was examined microscopically using a warm stage, X400 magnification and phase contrast optics. The proportion (%) of cells showing residual activity was determined. For semen processed in skim milk, egg yolk and glycerol diluent (SMEG) (Table 2 ), the contents of one 0.25 ml French straw were added to 1 ml of test solution with an osmolarity of 100 mOsm kg- 1. Incubation and microscopy were carded out as per raw semen. The methods described above were developed by examining the effects of variation in the method of reading, osmolarity and incubation time. For 77 tests of raw semen and 75 tests post-thaw, the proportion of cells showTable 1 Osmotic resistance test solution formulae

Fructose (g) Trisodium citrate (g) Distilled or R o / D I water to Osmolarity (mOsm kg- l )

Raw semen test

Post-thaw semen test

13.51 7.35 1000 ml 150

9.0 4.9 1000 ml 100

RolDI, reverse osmosis/ion purified.

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79

ca~( ~ 3

t--.--

J

Fig. 1. Types of swollen spermatozoa seen in the osmotic resistance test. (a) Non-swollen tail; (b) activity not seen. Judged by staining reaction or normal/abnormal acrosome. The arrow indicates the point where activity is most commonly seen. Table 2 Freezing diluent formula UHT skim milk (%) Glycerol (%) Egg yolk (%) Fructose (g%) Benzylpenicillin sodium (IU 1-1 ) Streptomycin base (mg 1-1 ) Lincomycin (mg 1-~ ) Spectinomycin (mg !-l )

83 7 10 1.25 500000 500 222 444

ing swelling irrespective of activity was determined and the result compared with the proportion showing activity. The latter method was adopted for all subsequent work. Five ejaculates from five bulls were tested raw in solutions of 150, 75 and 37.5 mOsm kg- i. Five further ejaculates, processed in SMEG diluent, were tested after freezing and thawing using test solutions of 125, 100, 75 and 50 mOsm kg -l. Ten ejaculates from ten bulls were incubated in 150 mOsm kg-1 test solution and results obtained after 20, 40, 60, 80 and 100 min incubation at 35°C. An identical experiment was carded out using ten ejaculates frozen and thawed in SMEG diluent. The raw semen was mixed well and two ORT preparations were made from each sample. Four counts of 100 sperm were performed from each preparation. In the case of post-thaw semen, the contents of each straw were emptied into a polystyrene tube, mixed well and two ORT preparations made with a semen:test solution ratio equal to that produced by emptying one straw into 1 ml

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of solution. Again, four counts of 100 sperm were made from each preparation. Repeatability was estimated as an intra-class correlation from between sample (ejaculate for raw and straw for frozen) and within sample analysis after adjusting for the effects of preparation, using the VARCOMP procedure of the Statistical Analysis Systems Institute (SAS, 1988 ). In order to study sources of variation, data on 401 ejaculates from 80 bulls were analysed using the NESTED procedure (SAS, 1988 ). The number of ejaculates per bull ranged from three to nine. Three straws per ejaculate were examined, a count of 100 sperm being made in each case. Initially, the relationship between the ORT result and fertility was measured by the 49-day non-return rate (NRR) in a group of seven yearling Friesian/Holstein bulls. Progeny test inseminations were carried out using five or six ejaculates from these young bulls. Each ejaculate conformed to a morphological standard of less than 15% total abnormal spermatozoa, with no single defect exceeding 5%. These ejaculates were submitted to the ORT (three straws per ejaculate). The semen was then divided into two groups according to ORT result. The ORT values of groups reflected the range of results obtained from individual bulls. Thus, the within-bull groups were widely separated for some individuals and less so for others. The two groups from each bull were sent to different AI Centre areas, this being the only convenient way at that time of obtaining separate group NRRs. A mean of 251 inseminations was made per bull (range 172-378). This study was then continued until a total of 48 bulls had been included.

3. Results

3.1. Method of reading For 77 ejaculates tested in the raw state, the proportion of active cells ranged from 17 to 82%, and inactive, swollen cells from I to 22%. These inactive cells accounted for between 1.5 and 91.7% of all swollen sperm in individual samples. In the case of 75 ejaculates tested post-thaw, active cells ranged from 13 to 54% and inactive from 9 to 46% with the latter forming from 19.5 to 68.2% of individual counts of total swollen cells. The mean ( + SD) percentage of inactive, swollen cells for raw and post-thaw tests was 5.5 _ 4.2 and 25.1 + 9.9, respectively. The increase seen in processed compared with raw samples suggests that diluent components formed a false semi-permeable membrane on some cells. When the results were expressed as the proportion of cells swollen, they were often well in excess of the percentage motile post-thaw. The proportion of cells showing activity was therefore adopted as the method to be used for all subsequent studies.

3.2. Osmolarity The response to varying osmolarity is shown in Table 3. The mean result for raw semen was highest at 150 mOsm kg- 1and this osmolarity also gave the great-

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Table 3 Effect of osmolarity on test result Raw semen Osmolarity (mOsm kg- ~) Mean test result ( n = 5 ) SD

Post-thaw semen Osmolarity (mOsm kg - ~) Mean test result ( n = 5 ) SD

150 48.2 23.8

75 14.0 11.2

37.5 1.0 1.4

125 30.6 6.6

100 30.6 5.9

75 19.2 7.8

50 13,6 4.9

Table 4 Effect of incubation time on test result Incubation time (min) 20

40

60

80

100

46.6 a

41.1 b

40.6 b

38.9 b

36.5 b

44.0 a

40.4 b

38.3 b

34.9 c

28.5 d

Raw semen mean test result ( n = 10)

Post-thaw semen mean test result ( n = 10)

Within each class of test, means with the same superscript are not significantly different at the 5% level of probability (Duncan's multiple range test).

est range of result, thus giving better means of discrimination between ejaculates. In solutions ofosmolarity greater than 150 mOsm kg- 1, sperm were still progressively motile and therefore could not be counted accurately. Below 150 mOsm kg- 1, a large proportion of the sperm was rendered immotile. For processed semen, a lower osmolarity was used to take account of the substantial volume of high osmolarity diluent present. Although the results for 125 and 100 mOsm kg- 1 were very similar, the latter was selected as standard since there was still some progressive motility present at the former which affected the ease and accuracy of counting.

3.3. Incubation time The effect of time on the test result is shown in Table 4. There was an initial period of change demonstrated by the significant (P< 0.05 ) difference in the results at 20 and 40 min incubation for both raw and post-thaw semen. No significant difference between the results was found at 40, 60, 80 and 100 rain for raw semen, and 40 and 60 min for post-thaw semen. A protocol was adopted of reading all tests after a minimum of 40 and maximum of 60 min incubation.

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Table 5 Sources of variation (post-thaw semen) Source

d.f.

MS

Variance component

Percent of total

Bulls Batches within bulls Straws within batches

80 320 802

1321.58 175.12 22.79

77.22 50.78 22.79

51.21 33.68 15.11

3.4. Repeatability The repeatability of the test for raw semen was 0.85 and for post-thaw semen 0.93.

3.5. Range The range of post-thaw ORT results over 401 ejaculates from 80 bulls was very wide, extending from 11 to 78%. Raw semen results for 307 ejaculates from 63 bulls ranged from 11 to 83%.

3.6. Sources of variation The relative importance of the effects of bull, batch (ejaculate) and straw within batch is shown in Table 5.

3.7. Relationship to fertility In the group of 48 bulls, raw ORT correlated reasonably well with post-thaw ORT (r=0.56) but not with NRR (r=0.08). Post-thaw ORT results for the initial group of seven bulls gave a correlation with fertility of 0.6 ( P < 0.01 ). Over the whole series of 48 bulls, this was reduced to 0.37. However, after adjusting the NRR for differences between bulls (GLM procedure; SAS, 1988 ), a correlation of 0.79 was obtained.

4. Discussion Up to the early 1960s, sperm were considered to be incapable of swelling, but swelling was subsequently demonstrated by Drevius and Eriksson (1966). The proportion of swollen bovine sperm in hypotonic saline was found to correlate almost significantly with the 60-90 day NRR, using fresh semen, by Bredderman and Foote (1969). Tail deformations in swollen bull spermatozoa were illustrated by Drevius (1972). Jeyendran et at. (1984), working with human semen, developed a hypo-os-

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motic swelling test (HOS) using a 150 mOsm kg-1 fructose/trisodium citrate test solution. This gave a very high correlation ( r = 0.94 ) with the denuded hamster oocyte penetration test (HEPT). This test solution was adopted for use in the present study, with the osmolarity adjusted according to the nature of the sample, i.e. raw semen or semen in diluent. An optimum osmolarity for each type of test was demonstrated by the use of several strengths of test solution. At osmolarities above the optimum, progressively motile sperm were present, which makes the test difficult to count; lower osmolarities resulted in rupture of many cells and loss of activity in others, reducing the range of results and thereby the grounds for discrimination between ejaculates. Jeyendran et al. (1984) expressed the result of their test as the percentage of cells showing swelling. In the present study, it was noted that in the case of bull spermatozoa there was a distinct subpopulation of these cells showing residual activity. In many cases, the total percentage of swollen cells gave a result, particularly in processed semen, considerably in excess of the population of motile sperm present in the untreated sample. Consequently, separate counts of active and inactive swollen sperm were made initially. Recently, the use of the fluorescent stain H33258 (De Leeuw et al., 1991 ) in conjunction with the test resulted in inactive swollen cells showing fluorescence, thus confirming their non-viability (S.G. Revell and R.M. Hating, unpublished observations, 1992). The decision to limit the test result to the count of active cells only would therefore appear to have been correct. The use of the HOS test in the human field has received something of a mixed press. For HEPT, it has been found to be predictive (Okada et al., 1990), nonpredictive (Chan et al., 1988) or no better than other assessments (Rogers and Parker, 1991 ). The test has been applied successfully for in vitro fertilisation (IVF) by Van der Ven et al. (1986) and Daya et al. ( 1989 ), while others (Barratt et al., 1989; Avery et al., 1990; Chan et al., 1990) found it unhelpful in predicting IVF success. A neutral conclusion was drawn by Liu et al. (1988) and Sjoblom and Coccia (1989). The HOS does appear to be an indicator of male factor infertility in humans (Bernt et al., 1989), infertile males being associated with a result of less than 50% (Check et al., 1989; Coetzee et al., 1989). Of 1890 pre-vasectomy ejaculates from previously fertile men, 95% showed an HOS greater than 60% (De Castro et al., 1990). An ORT for boar semen was developed by Schilling and Vengust ( 1985 ), using acrosome morphology. Each sample was examined after 15 min incubation at 39°C in an isotonic medium and after 120 min incubation at 150 mOsm kg-1. The percentage of sperm showing a normal apical ridge (NAR) was counted in each case and the result taken as the sum of the two results divided by 2. High correlations between boars ( n = 34) were noted for ORT vs. NAR and motility both post-thaw and after 72 h storage (range 0.59-0.81 ). When boars were grouped by ORT (high, medium and low), NAR and motility in the high and low groups were deafly separated, but the medium group overlapped both considerably. When ejaculates were ranked into five groups by ORT, mean NAR and motility again followed the rankings. Increased frequency of ejaculation in boars from

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twice per week to alternate days reduced ORT and motility after 72 h storage (Schilling and Vengust, 1987). ORT was recommended as a useful quality control technique. When sows were inseminated using semen from 67 boars ranked by ORT (less than 56, 57-68, over 69), the farrowing rates were 70.4, 70.8 and 81.3, respectively, with litter sizes of 8.2, 9.2 and 10.2 (Schilling, 1989). It was decided to use the tail swelling response to hypotonicity as the means of assessing bovine semen as it is more easily seen irrespective of the orientation of the sperm head to the observer. The test can therefore be performed more rapidly than one based on acrosome assessment. This is most important in a busy commercial laboratory where large numbers of samples are processed. The test gives clear, repeatable results over a wide range which allows better discrimination between ejaculates than the subjective motility assessments currently used for quality control. Results from the latter tend to be expressed as values close to the pass mark with little differentiation of quality between batches which pass the test. The NRRs used in the present study were uncorrected for factors such as technician, area and parity of cow. Indeed, the means of obtaining separate NRRs for high and low ORT semen from each bull was to use them in different areas. However, the areas covered were extensive and involved large numbers of inseminatots. Each area was given similar numbers of high and low batches in the course of the experiment to reduce the effect of area and technician. Young progeny test bulls were used because of the practice of using semen quickly, in only one breed of cow, from a minimum of five ejaculates and on account of the trial semen being the only semen available from such bulls. This system also provided a regular supply of fresh bulls but the number of inseminations was rather limited, especially in cases where one 'group' consisted of only one ejaculate and the other of four or five. Semen from the initial group of seven bulls from this trial was subjected to computer-aided sperm motility analysis using a Hamilton-Thorn 2030 apparatus (L. Robertson, S.Y. Middleton and S.G. Revell, unpublished data, 1993). The motility characteristic which correlated most closely with ORT was amplitude of lateral head displacement (ALH) (0.67, P < 0.001 ). The best 49-day NRR correlation with movement was that with ALH ( r = 0.43 ). Post-thaw ORT (reported above) showed a better relationship to NRR. In the whole series of 48 bulls, those showing ejaculate ORT results consistently in excess of 38% were invariably also above the mean NRR. Commercial considerations require maximum production from high demand bulls. This can best be achieved by manipulation of the dose, which must be done without compromising fertility. Normal levels of fertility can be achieved at doses much lower than the 20 million total sperm currently used routinely in this laboratory (Den Daas, 1992; S.G. Revell, unpublished observations, 1977 ). The post-thaw ORT shows promise as a practical routine quality control procedure which could also be used to support dose reduction, initially by identifying high NRR/high ORT bulls and then by assessment of batches frozen at reduced dose, with a high pass level (at least 38%) being set. The very high correlation (0.79) achieved between NRR and post-thaw ORT after removal of bull effects indicates that whilst unexplained differences in fertility between bulls are present, in the majority of cases ORT

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used within bull will rank ejaculates correctly for fertility. Ejaculate and straw effects were the sources of a great deal of variation in ORT seen in this study, which is consistent with earlier studies (Revell and Wood, 1978 ). It is therefore essential to assess individual batches by the examination of a number of straws from each.

Acknowledgements I am indebted to Tecwyn Roberts and Kay Ellams for technical assistance and Elaine Lloyd for secretarial support. My thanks are also due to Paul Watson and Peter Merson for reading the manuscript.

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